Heavy Metal Binding Compounds and Their Method of Use

ABSTRACT

The present invention provides a metal chelator and methods that facilitate binding, detecting, monitoring and quantitating of heavy metal ions in a sample. This metal chelating moiety is a —N,N,O-triacetic acid analog of BAPTA and has the following formula

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 12/397,896, filed Mar. 4, 2009, which is adivisional application of U.S. patent application Ser. No. 11/622,900,filed Jan. 12, 2007, now U.S. Pat. No. 7,521,577, which claims priorityto U.S. Provisional Patent Application No. 60/758,830, filed Jan. 12,2006, the disclosures of which are hereby incorporated herein byreference in their entirety as if set forth fully herein.

FIELD OF THE INVENTION

The present invention relates to novel compositions and methods for thedetection of heavy metal ions. The invention has applications in thefields of cell biology, neurology, nutrition, immunology, reproductivebiology, cancer and proteomics.

BACKGROUND OF THE INVENTION

Certain transition and heavy metal ions pose increasing environmentaland health risks. For example, as the use of Ni—Cd batteries increases,so does the prevalence of nickel and cadmium ions in manufacturing,disposal, and environmental contamination. It has long been known thatmercury ions are a persistent and prevalent health risk, with a largepercentage of the populace exposed to the risk. The same goes for leadions, found in peeling paint on older buildings. These increasingincidences result in increasing exposures and internalization of theseions within individuals.

Thus there exists a need for increasingly sophisticated methods for thedetection and quantitation of certain heavy metal and transition metalions in a variety of samples, ranging from groundwater and soil toinside human cells. In the biomedical research field, luminescence-basedprobes of alkaline earth metal cations such as calcium have been ofenormous benefit. To date there have been some examples ofluminescence-based detection methods developed for heavy metal andtransition metal ions. However these existing methods rely uponsubstandard compounds which lack specificity, dynamic range,sensitivity, and applicability to field use. The present inventionaddresses these shortcomings by describing novel luminescence-basedmaterials that are very useful for the detection and quantitation ofcertain metal ions such as cadmium(II), lead(II), mercury(II), andnickel(II).

SUMMARY OF THE INVENTION

Embodiments of the present invention provide metal ion reportercompounds according to the formula:

R_(X), R_(y) and R_(x)′ are each independently is hydrogen, alkyl,substituted alkyl or —CR¹³R¹⁴CO₂R, wherein R is H, a salt ion or—CH₂OC(O)(CH₂)_(n)CH₃ and n is 0 to 6. Each R¹³ and R¹⁴ is independentlyhydrogen, alkyl or substituted alkyl.

R¹-R⁸ are independently hydrogen, halogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, hydroxyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, amino, substituted amino,alkylamino, substituted alkylamino, aldehyde, carboxyl, azido, nitro,nitroso, cyano, thioether, sulfoalkyl, carboxyalkyl, aminoalkyl,reporter molecule, reactive group, carrier molecule or solid support.Alternatively, any adjacent R¹-R⁸ together with the atoms to which theyare joined, form a ring which is a 5-, 6- or 7-memberedheterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-memberedcycloalkyl, a 5-, 6- or 7-membered heteroaryl, a substituted 5-, 6- or7-membered heteroaryl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6-or 7-membered aryl or reporter molecule.

The bridge substituents R⁹-R¹² are independently hydrogen, alkyl,substituted alkyl, reactive group, carrier molecule, or solid support.Alternatively, R⁹ in combination with R¹⁰; or R¹¹ in combination withR¹² together with the atoms to which they are joined, form a ring whichis a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or7-membered heterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, asubstituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheteroaryl, a substituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or7-membered aryl, or a substituted 5-, 6- or 7-membered aryl.

In one embodiment, the present compound comprises a reporter moleculethat is a chromophore, fluorophore, fluorescent protein, phosphorescentdye or a tandem dye. In one aspect, the reporter molecule is a xanthene,indole, cyanine, oxazole, dansyl, borapolyazaindacene, benzofuran,quinazolinone, benzazole, oxazine, pyrene, naphthalene, coumarin,biotin, enzyme substrate or fluorescent protein. In a further aspect,the xanthene is a fluorescein or derivative thereof, rhodamine orderivative thereof, rhodol or derivative thereof or rosamine or aderivative thereof. In another further aspect, the reporter molecule isoptionally and independently substituted by hydrogen, halogen, amino,substituted amino, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, sulfo, lipophilic group (AMester or acetate ester) solid support, reactive group or carriermolecule.

Additional embodiments of the present invention provide methods ofdetecting the presence or absence of metal ions in a sample. The presentmethods comprise:

-   -   a. combining a present metal ion reporter molecule with the        sample to prepare a labeling mixture;    -   b. incubating the labeling mixture for a sufficient amount of        time for the metal ion reporter molecule to associate with metal        ions in the sample to form an incubated mixture;    -   c. illuminating the incubated sample with an appropriate        wavelength to form an illuminated mixture; and,    -   d. observing the illuminated mixture whereby the presence or        absence of the metal ions in a sample is detected.

Also provided is a staining solution comprising a present compound andan aqueous buffer solution. In one aspect the aqueous buffer solution isnormal saline, PBS, cell culture media, MOPS, Good's buffer or PIPESbuffer.

Further embodiments provide complexes of the present compoundsnon-covalently associated with metal ions and compositions comprising apresent compound and a sample. In one aspect the sample comprises livingcells, cellular components, proteins, peptides, buffer solutions,intracellular fluids, extracellular fluids, fixed cells, biologicalfermentation media, environmental sample, industrial samples or chemicalreactors, eukaryotic cells, or prokaryotic cells. In a further aspect,the sample comprises blood cells, immune cells, cultured cells, muscletissue, neurons, extracellular vesicles; vascular tissue, blood fluids,saliva, urine; water, soil, waste water, sea water; pharmaceuticals,foodstuffs or beverages.

Additional embodiments of the present invention provide kits for thedetection of metal ions, wherein the kit comprises any compound of thepresent invention. In a further embodiment, the kits compriseinstructions for detecting the presence or absence of lead, mercury,nickel, lanthanum or cadmium ions in a sample, in particular areincluded instructions for detecting the presence or absence of lead,mercury, nickel, lanthanum or cadmium ions in a sample using flowcytometry. In yet another further aspect, the kits comprises at leastone component that is a sample preparation reagent, a buffering agent,aqueous metal ion reporter molecule dilution buffer, an additionaldetection reagent, a metal ion calibration reagent, a positive control,a metal ion indicator other than for lead, mercury, nickel, lanthanum orcadmium ions, an antibody or fragment thereof or a reference dyestandard.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Shows the detection and titration of A) lead ions (0 to 50 μM)(Kd=2.1 μM), B) cadmium ions (0 to 100 μM) (Kd=4.6 μM) and C) mercuryions (0 to 100 μM) (Kd=78 μM) in a solution containing MOPS (pH 7.0) and1 μM of Compound 15. Excitation 550 nm.

FIG. 2: Shows the detection and titration (0 to 100 μM) of A) lead ions(Kd=4.9 μM), B) cadmium ions (Kd=1.3 μM) and C) mercury ions (Kd=75 μM)in a solution containing 50 mM MOPS (pH 7.0) and 1 μM of Compound 10.Excitation 492 nm.

FIG. 3: Shows the detection and titration of cadmium ions (0 to 20 μM)(Kd=1.2 μM) in a solution containing 50 mM MOPS (pH 7.0) and 1 μM ofCompound 18. Excitation 348 nm.

FIG. 4: Shows the detection of intracellular lead ions (I μM PbCl₂ addedto Jurkat cells in the presence of an ionophore) with Compound 12.

FIG. 5: Shows the change in fluorescence intensity based onconcentration of PbCl₂ (1 nM to 1 μM) added to Jurkat cells, 5a) is anoverlay of histograms showing how concentration of PbCl₂ affects thefluorescence and 5b) is a plot of Mean Fluorescence Intensity (MFI)versus PbCl₂ concentration that shows increasing fluorescence withincreasing PbCl₂ concentration.

FIG. 6: Shows that Compound 12 stains only viable cells (when incubatedwith PbCl₂ in the presence of an ionophore) without causing cell deathwherein 6a) is a dot plot of forward scatter vs side scatter with twopopulations of cells, wherein the group of cells on the left are dead(stained with Propidium Iodide (PI)) while the cells on the right arestained with Compound 12 (viable cells); 6b) is the fluorescence fromCompound 12 (incubated with lead chloride solution), collected at525/20BP; 6c) is the fluorescence from Propidium Iodide, collected at610/20BP; and 6d) is a dual color fluorescence plot (525BP vs 610BP)wherein cells in the top left quadrant are positive for PI (dead cells)while the cells in the bottom right quadrant are positive for Compound12 (live cells).

FIG. 7: Shows the histogram from cells incubated with Compound 12 andfree calcium ions demonstrating that Compound 12 does not detectintracellular calcium ions.

FIG. 8: Shows the detection of intracellular cadmium ions (250 nM to 1μM added to Jurkat cells in the presence of an ionophore) with Compound12.

FIG. 9: Shows that Compound 12 stains only viable cells (when incubatedwith CdCl₂ in the presence of an ionophore) without causing cell deathwherein 9a) is the fluorescence from Compound 12 (incubated with cadmiumchloride solution), collected at 525/20BP; 9b) is the fluorescence fromPropidium Iodide (PI), collected at 610/20BP; 9c) is a dual colorfluorescence plot (525BP vs 610BP) wherein the cells on the bottom rightquadrant are positive for Compound 12 (live cells incubated with CdCl₂solution) while the cells in the top left quadrant are positive for PI(dead cells).

FIG. 10: Shows the change in fluorescence intensity of Compound 12 basedon concentration of CdCl₂ (5 μM to 500 μM) added to cells as a plot ofMean Fluorescence Intensity (MFI) versus CdCl₂ concentration wherein theMFI increases with increasing PbCl₂ concentration.

FIG. 11: Shows the detection (MFI=57.3) of intracellular lead ions (1 μMPbCl2 and 1 μM ionomycin) in human red blood cells with Compound 12 (500nM) compared to negative controls (MFI=2.6-13.6).

FIG. 12: Shows that the fluorescent signal is due to intracellular Pb orCd ions respectively. FIG. 12 a) shows five histograms (0, 125 nM, 250nM, 500 nM and 1 μM of PbCl₂) of the detection of intracellular leadions wherein the cells in the top section are the dead cells (PropidiumIodide) and the cells on the bottom section are the live cells stainedwith Compound 12 (400 nM); 12b) is three dual color labeled plots,wherein panel A is background fluorescence from live and dead cells,Panel B is cells incubated with Compound 12 (400 nM), ionomycin (1 μM)and PbCl₂ (500 nM) vs PI and Panel C is Panel B cells incubated with theintracellular metal chelator TPEN, the drop in fluorescence from theviable cells (cells on the bottom section) demonstrates that thefluorescence is due to the lead chloride in the cells; 12c) shows fourhistograms (0, 25 uM, 50 μM and100 μM of CdCl₂); 12d) is three dualcolor labeled plots are shown, wherein Panel A is backgroundfluorescence from live and dead cells, Panel B is cells incubated with400 nM Compound 12, 100 μM CdCl₂ and 1 μM ionomycin vs PI and Panel C isPanel B cells incubated with the intracellular metal chelator TPEN, thedrop in fluorescence from the viable cells (cells on the bottom section)demonstrates that the fluorescence is due to the cadmium chloride in thecells.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present invention provides heavy metal-binding compounds for thebinding, including sequestering of ions, detection, monitoring andquantification of heavy metal ions, including physiologicalconcentrations of heavy metal ions that are present in intracellular andextracellular biological fluids. The heavy metal-binding compoundspreferentially bind heavy metal ions (lead, cadmium, mercury, nickel,and/or lanthanum) in the presence of physiological concentrations ofcalcium ions, this property being a function of the chelating moiety ofthe heavy metal-binding compounds, and provides a mechanism for bindingand detecting heavy metal ions in the presence of calcium ions.

Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise.

Thus, for example, reference to “a present compound” includes aplurality of compounds and reference to “a metal ion” includes aplurality of ions and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for purposes of the invention as described herein.

The term “affinity” as used herein refers to the strength of the bindinginteraction of two molecules, such as a metal chelating compound and ametal ion or a positively charged moiety and a negatively chargedmoiety.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds of the invention may be prepared as a single isomer (e.g.,enantiomer, cis-trans, positional, diastereomer) or as a mixture ofisomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose andappropriate method for a particular situation. See, generally, Furnisset al. (eds.), VOGEL's ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY5^(TH) ED., Longman Scientific and Technical Ltd., Essex, 1991, pp.809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “acyl” or “alkanoyl” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof,consisting of the stated number of carbon atoms and an acyl radical onat least one terminus of the alkane radical. The “acyl radical” is thegroup derived from a carboxylic acid by removing the —OH moietytherefrom.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include divalent(“alkylene”) and multivalent radicals, having the number of carbon atomsdesignated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”.

Exemplary alkyl groups of use in the present invention contain betweenabout one and about twenty-five carbon atoms (e.g. methyl, ethyl and thelike). Straight, branched or cyclic hydrocarbon chains having eight orfewer carbon atoms will also be referred to herein as “lower alkyl”. Inaddition, the term “alkyl” as used herein further includes one or moresubstitutions at one or more carbon atoms of the hydrocarbon chainfragment.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cycliccarbon-containing radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si, P and S, and wherein the nitrogen,phosphorous and sulfur atoms are optionally oxidized, and the nitrogenheteroatom is optionally be quaternized. The heteroatom(s) O, N, P, Sand Si may be placed at any interior position of the heteroalkyl groupor at the position at which the alkyl group is attached to the remainderof the molecule. Examples include, but are not limited to,—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may beconsecutive, such as, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.Similarly, the term “heteroalkylene” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic moiety that can be a single ring or multiple rings (preferablyfrom 1 to 3 rings), which are fused together or linked covalently.

The term “heteroaryl” as used herein refers to an aryl group as definedabove in which one or more carbon atoms have been replaced by anon-carbon atom, especially nitrogen, oxygen, or sulfur. For example,but not as a limitation, such groups include furyl, tetrahydrofuryl,pyrrolyl, pyrrolidinyl, thienyl, tetrahydrothienyl, oxazolyl,isoxazolyl, triazolyl, thiazolyl, isothiazolyl, pyrazolyl,pyrazolidinyl, oxadiazolyl, thiadiazolyl, imidazolyl, imidazolinyl,pyridyl, pyridaziyl, triazinyl, piperidinyl, morpholinyl,thiomorpholinyl, pyrazinyl, piperainyl, pyrimidinyl, naphthyridinyl,benzofuranyl, benzothienyl, indolyl, indolinyl, indolizinyl, indazolyl,quinolizinyl, qunolinyl, isoquinolinyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, pteridinyl, quinuclidinyl, carbazolyl,acridinyl, phenazinyl, phenothizinyl, phenoxazinyl, purinyl,benzimidazolyl and benzthiazolyl and their aromatic ring-fused analogs.Many fluorophores are comprised of heteroaryl groups and include,without limitations, xanthenes, oxazines, benzazolium derivatives(including cyanines and carbocyanines), borapolyazaindacenes,benzofurans, indoles and quinazolones.

The above heterocyclic groups may further include one or moresubstituents at one or more carbon and/or non-carbon atoms of theheteroaryl group, e.g., alkyl; aryl; heterocycle; halogen; nitro; cyano;hydroxyl, alkoxyl or aryloxyl; thio or mercapto, alkyl- or arylthio;amino, alkyl-, aryl-, dialkyl-, diaryl-, or arylalkylamino;aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl or arylalkylaminocarbonyl;carboxyl, or alkyl- or aryloxycarbonyl; aldehyde; aryl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- orarylsulfonyl; hydroximinyl, or aryl- or alkoximinyl. In addition, two ormore alkyl substituents may be combined to form fused heterocycle-alkylring systems. Substituents including heterocyclic groups (e.g.,heteroaryloxy, and heteroaralkylthio) are defined by analogy to theabove-described terms.

The term “heterocycloalkyl” as used herein refers to a heterocycle groupthat is joined to a parent structure by one or more alkyl groups asdescribed above, e.g., 2-piperidylmethyl, and the like. The term“heterocycloalkyl” refers to a heteroaryl group that is joined to aparent structure by one or more alkyl groups as described above, e.g.,2-thienylmethyl, and the like.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ′NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. In theschemes that follow, the symbol X represents “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)-U-, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S), phosphorus (P) and silicon (Si).

The term “amino” or “amine group” refers to the group —NR′R″ (or NRR′R″)where R, R′ and R″ are independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl. Asubstituted amine being an amine group wherein R′ or R″ is other thanhydrogen. In a primary amino group, both R′ and R″ are hydrogen, whereasin a secondary amino group, either, but not both, R′ or R″ is hydrogen.In addition, the terms “amine” and “amino” can include protonated andquaternized versions of nitrogen, comprising the group —NRR′R″ and itsbiologically compatible anionic counterions.

The term “aqueous solution” as used herein refers to a solution that ispredominantly water and retains the solution characteristics of water.Where the aqueous solution contains solvents in addition to water, wateris typically the predominant solvent.

The term “biotin” as used herein refers to any biotin derivative,including without limitation, substituted and unsubstituted biotin, andanalogs and derivatives thereof, as well as substituted andunsubstituted derivatives of caproylamidobiotin, biocytin,desthiobiotin, desthiobiocytin, iminobiotin, and biotin sulfone.

The term “buffer” as used herein refers to a system that acts tominimize the change in acidity or basicity of the solution againstaddition or depletion of chemical substances.

The term “carbonyl” as used herein refers to the functional group—(C═O)—. However, it will be appreciated that this group may be replacedwith other well-known groups that have similar electronic and/or stericcharacter, such as thiocarbonyl (—(C═S)—); sulfinyl (—S(O)—); sulfonyl(—SO₂)—), phosphonyl (—PO₂—).

The term “carboxy” or “carboxyl” refers to the group —R′(COOR) where R′is alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroaryl, or substituted heteroaryl. R ishydrogen, a salt or —CH₂OC(O)CH₃.

The term “carrier molecule” as used herein refers to a biological or anon-biological component that s covalently bonded to compound of thepresent invention. Such components include, but are not limited to, anamino acid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell, a virus and combinationsthereof.

The term “cell permeable” as used herein refers to compounds of thepresent invention that are able to cross the cell membrane of livecells. Lipophilc groups that are covalently attached to the presentcompounds facilitate this permeability and live cell entry. Once insidethe cells, the lipophilic groups are hydrolyzed resulting in chargedmolecules that are well retained in living cells. Particularly usefullipophilic groups include acetoxymethyl (AM) ester and acetate esterswherein once inside the cells the groups are cleaved by nonspecificesterases resulting in charged molecules.

The term “complex” as used herein refers to the association of two ormore molecules, usually by non-covalent bonding.

The term “detectable response” as used herein refers to a change in oran occurrence of, a signal that is directly or indirectly detectableeither by observation or by instrumentation. Typically, the detectableresponse is an optical response resulting in a change in the wavelengthdistribution patterns or intensity of absorbance or fluorescence or achange in light scatter, fluorescence lifetime, fluorescencepolarization, or a combination of the above parameters. Alternatively,the detectable response is an occurrence of a signal wherein thefluorophore is inherently fluorescent and does not produce a change insignal upon binding to a metal ion. Alternatively, the detectableresponse is the result of a signal, such as color, fluorescence,radioactivity or another physical property of the detectable labelbecoming spatially localized in a subset of a sample such as in a gel,on a blot, or an array, in a well of a micoplate, in a microfluidicchamber, or on a microparticle as the result of formation of a ternarycomplex of the invention that comprises a zinc binding protein.

The term “directly detectable” as used herein refers to the presence ofa detectable label or the signal generated from a detectable label thatis immediately detectable by observation, instrumentation, or filmwithout requiring chemical modifications or additional substances. Forexample, a fluorophore produces a directly detectable response.

The term “dye” as used herein refers to a compound that emits light toproduce an observable detectable signal. “Dye” includes fluorescent andnonfluorescent compounds that include without limitations pigments,fluorophores, chemiluminescent compounds, luminescent compounds andchromophores. The term “fluorophore” as used herein refers to a compoundthat is inherently fluorescent or demonstrates a change in fluorescenceupon binding to a biological compound or metal ion, i.e., fluorogenic.Numerous fluorophores are known to those skilled in the art and include,but are not limited to, coumarin, acridine, furan, indole, quinoline,cyanine, benzofuran, quinazolinone, benzazole, borapolyazaindacene andxanthenes, with the latter including fluoroscein, rhodamine, rhodol,rosamine and derivatives thereof as well as other fluorophores describedin RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBESAND RESEARCH CHEMICALS (9^(th) edition, CD-ROM, 2002).

The term “kit” as used herein refers to a packaged set of relatedcomponents, typically one or more compounds or compositions.

The term “Linker” or “L”, as used herein, refers to a single covalentbond or a series of stable covalent bonds incorporating 1-30 nonhydrogenatoms selected from the group consisting of C, N, O, S and P thatcovalently attach the phosphate-binding compounds to another moiety suchas a chemically reactive group or a phosphorylated target molecule.Exemplary linking members include a moiety that includes —C(O)NH—,—C(O)O—, —NH—, —S—, —O—, and the like. A “cleavable linker” is a linkerthat has one or more cleavable groups that may be broken by the resultof a reaction or condition. The term “cleavable group” refers to amoiety that allows for release of a portion, e.g., a reporter molecule,carrier molecule or solid support, of a conjugate from the remainder ofthe conjugate by cleaving a bond linking the released moiety to theremainder of the conjugate. Such cleavage is either chemical in nature,or enzymatically mediated. Exemplary enzymatically cleavable groupsinclude natural amino acids or peptide sequences that end with a naturalamino acid.

In addition to enzymatically cleavable groups, it is within the scope ofthe present invention to include one or more sites that are cleaved bythe action of an agent other than an enzyme. Exemplary non-enzymaticcleavage agents include, but are not limited to, acids, bases, light(e.g., nitrobenzyl derivatives, phenacyl groups, benzoin esters), andheat. Many cleaveable groups are known in the art. See, for example,Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshi et al.,J. Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J. Immunol.,124: 913-920 (1980); Bouizar et al., Eur. J. Biochem., 155: 141-147(1986); Park et al., J. Biol. Chem., 261: 205-210 (1986); Browning etal., J. Immunol., 143: 1859-1867 (1989). Moreover a broad range ofcleavable, bifunctional (both homo- and hetero-bifunctional) spacer armsare commercially available.

An exemplary cleavable group, an ester, is cleavable group that may becleaved by a reagent, e.g. sodium hydroxide, resulting in acarboxylate-containing fragment and a hydroxyl-containing product.

The linker can be used to attach the compound to another component of aconjugate, such as a targeting moiety (e.g., antibody, ligand,non-covalent protein-binding group, etc.), an analyte, a biomolecule, adrug and the like.

The term “metal chelator” or “metal chelating moiety” as used hereinrefers to a chemical compound that combines with a metal ion to form achelate ring structure. For the purposes of the present invention themetal chelator is an analog of BAPTA that has demonstrated affinity forlead, mercury, nickel, lanthanum, and cadmium. These ions may be free insolution or they may be sequestered by a metal ion-binding compound. Themetal chelators are optionally substituted by substituents that adjustthe ion-binding affinity, solubility, chemical reactivity, spectralproperties or other physical properties of the compound.

The term “metal ion” as used herein refers to any physiological,environmental and or nutritional relevant metal ion. Such metal ionsinclude certain transition metal ions and lanthanide metal ions, suchas, but are not limited to lead, mercury, nickel, lanthanum and cadmium.

The terms “protein” and “polypeptide” are used herein in a generic senseto include polymers of amino acid residues of any length. The term“peptide” is used herein to refer to polypeptides having less than 250amino acid residues, typically less than 100 amino acid residues, moretypically less than 15 amino acid residues. The terms apply to aminoacid polymers in which one or more amino acid residues are an artificialchemical analogue of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers. The peptide orprotein may be further conjugated to or complexed with other moietiessuch as dyes, haptens, radioactive isotopes, natural and syntheticpolymers (including microspheres), glass, metals and metallic particles,proteins and nucleic acids.

The term “reactive group” as used herein refers to a group that iscapable of reacting with another chemical group to form a covalent bond,i.e. is covalently reactive under suitable reaction conditions, andgenerally represents a point of attachment for another substance. Thereactive group is a moiety, such as a photoactivatable group, carboxylicacid or succinimidyl ester, on the compounds of the present inventionthat is capable of chemically reacting with a functional group on adifferent compound to form a covalent linkage resulting in aphosphate-binding labeled component. Reactive groups generally includenucleophiles, electrophiles and photoactivatable groups.

Exemplary reactive groups include, but not limited to, olefins,acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes,ketones, carboxylic acids, esters, amides, cyanates, isocyanates,thiocyanates, isothiocyanates, amines, hydrazines, hydrazones,hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides,disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids,acetals, ketals, anhydrides, sulfates, sulfenic acids isonitriles,amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamicacids thiohydroxamic acids, allenes, ortho esters, sulfites, enamines,ynamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates,imines, azides, azo compounds, azoxy compounds, and nitroso compounds.Reactive functional groups also include those used to preparebioconjugates, e.g., N-hydroxysuccinimide esters, maleimides and thelike. Methods to prepare each of these functional groups are well knownin the art and their application to or modification for a particularpurpose is within the ability of one of skill in the art (see, forexample, Sandler and Karo, eds. ORGANIC FUNCTIONAL GROUP PREPARATIONS,Academic Press, San Diego, 1989).

The term “reporter molecule” as used herein refers to a moiety that isused to facilitate detection of metal ions in combination with themetal-chelating moiety of the present invention. Illustrative reportermolecules include molecules that can be directly observed or measured orindirectly observed or measured such as fluorophores, radioactive,haptens, fluorescent proteins and enzyme reporter molecules (Patton, W.,et al, J. Chromatography B: Biomedical Applications (2002) 771:3-31;Patton, W., et al, Electrophoresis (2000) 21:1123-1144). Such reportermolecules include, but are not limited to, radiolabels that can bemeasured with radiation-counting devices; pigments, dyes (fluorophore orchromophore) or other chromogens that can be visually observed ormeasured with a spectrophotometer; tandem dyes that participate inenergy transfer, spin labels that can be measured with a spin labelanalyzer; and fluorescent proteins or fluorophores, where the outputsignal is generated by the excitation of a suitable molecular adduct andthat can be visualized by excitation with light that is absorbed by thedye or can be measured with standard fluorometers or imaging systems,for example or metal particles, e.g. gold or silver particles ormetallic bar codes that can be detected by their optical orlight-scattering properties. The reporter molecule can be achemiluminescent substance, where the output signal is generated bychemical modification of the signal compound; a metal-containingsubstance; or an enzyme, where there occurs an enzyme-dependentsecondary generation of signal, such as the formation of a coloredproduct from a colorless substrate. The term reporter molecule can alsorefer to a “tag”, hapten or other ligand that can bind selectively to alabeled molecule such that the labeled molecule, when addedsubsequently, is used to generate a detectable signal. For example, onecan use biotin as a tag and then use an avidin or streptavidin conjugateof horseradish peroxidase (HRP) to bind to the tag, and then use achromogenic substrate (e.g., tetramethylbenzidine) or a fluorogenicsubstrate such as Amplex® Red reagent (Molecular Probes, Inc.) to detectthe presence of HRP. Numerous reporter molecules and tags and methodsfor their selective detection are known by those of skill in the art andinclude, but are not limited to, particles, fluorophores, haptens,enzymes and their chromogenic, fluorogenic and chemiluminescentsubstrates and other labels that are described in the MOLECULAR PROBESHANDBOOK, supra. In addition, present reporter molecules can besubstituted with substituents that alter the ion-binding affinity of thepresent compound, solubility, chemical reactivity, spectral propertiesor other physical properties of the reporter molecule.

The term “sample” as used herein refers to any material that may containmetal ions, as defined above. Typically, the sample is a live cell or abiological fluid that comprises endogenous host cell proteins orfoodstuff or an environmental sample such as a water sample. The samplemay be in an aqueous solution, a viable cell culture or immobilized on asolid or semi solid surface such as a polyacrylamide gel, membrane blotor on a microarray.

The term “metal ion reporter molecule” or “present compound” as usedherein refers to the metal chelating moiety that is an analog of BAPTA.

These compounds are typically substituted by substituents that modifyion affinity, chemical reactivity, spectral properties and solubility.Typically, the present compound is substituted by a reporter molecule,reactive group, solid support or carrier molecule that facilitatebinding, detection, isolation, sequestration, and monitoring of thepresent metal ions. Preferably, the present compounds are substituted byreporter molecules that facilitate detection and monitoring of the metalions. Thus, the metal ion reporter compounds of the present inventiontypically have the general formula A(B) wherein A is a reportermolecule, reactive group or carrier molecule, B is a metal chelatingmoiety. The reporter molecule may share atoms of the chelating moiety,be attached by a single covalent bond or attached by a linker comprisingmultiple stable bonds. Reactive groups and carrier molecules areattached by a single covalent bond or by a linker comprising multiplestable bonds. Thus, when these substituents are attached by a singlecovalent bond or a series of stable bonds the metal ion binding compoundhas the general formula A(L)m(B) wherein L is a Linker that covalentlyattaches the substituents to the metal chelating moiety and m is 0-4.The metal ions, such as lead and cadmium ions can be free in solutionsor non-covalently bound to another molecule such as a protein.

The Compounds

In general, for ease of understanding the present invention, the heavymetal-binding compounds and corresponding substituents will first bedescribed in detail, followed by the many and varied methods in whichthe heavy metal-binding compounds and metal ions find uses, which isfollowed by exemplified methods of use and synthesis of certain novelcompounds that are particularly advantageous for use with the methods ofthe present invention.

The present invention provides analogs of the metal ion chelator, BAPTA,that bind a wide range of metal cations including physiological relevantlevels of metal cations such as lead, cadmium and mercury. These metalchelating compounds comprise substituents well known in the artincluding linkers, chemically reactive groups, carrier molecules, solidsupport and reporter groups.

The present compounds find utility in binding target metal ions in asample. The sample includes live cells or a biological fluid thatcomprises endogenous host cell proteins, buffer solutions andenvironmental samples. Therefore, when the present metal ion-bindingcompound comprises a reporter molecule they find utility inquantitating, monitoring and detecting target metal ions. Thesecompounds are herein referred to as metal ion reporter molecules.Typically, the reporter molecule is directly attached to the benzomoiety or two of the benzo substituents when taken in combination form afused reporter molecule. Detection of target metal ions can also beaccomplished in live cells wherein the present compound comprises alipophilic group such as an AM or acetate ester that allows for entryacross the live cell membrane. Once inside the cells nonspecificesterases cleave the AM or acetate ester resulting a charged moleculethat is well retained in the cell. These present compounds areparticularly useful for binding physiological relevant levels of lead,cadmium and mercury ions.

Chelating Moiety

The metal chelating moiety of the present invention is a derivative ofthe well known calcium chelating moiety1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). In aneffort to devise a lead, cadmium, nickel, and/or mercury probe that isselective for physiologically relevant levels of these ions but notcalcium ions, which are typically present in much higher concentrations,we unexpectedly found that by replacing one of the nitrogen atoms withoxygen, resulting in a triacetic BAPTA analog, that the new compoundcould selectively detect physiological levels of lead, cadmium andmercury, but not calcium ions. The modification to the BAPTA chelatingmoiety reduced the affinity for calcium ions by approximately 100-1000×fold but not the affinity for lead ions resulting in a chelator thatpreferentially binds lead ions in the presence of physiologicalconcentrations of calcium ions. This metal chelating moiety is a—N,N,O-triacetic acid analog of BAPTA and has the following formula:

R_(X), R_(Y), and R_(X) are independently hydrogen, acetic acid or anacetic acid group that has been substituted by an acetyloxy methyl (AM)ester wherein acetic acid is represented by —CR¹³R¹⁴CO₂R wherein R ishydrogen, a salt ion or an AM ester represented by —CH₂OC(O)(CH₂)_(n)CH₃and n is 0 to 6. R¹³ and R¹⁴ are independently hydrogen, alkyl orsubstituted alkyl. In one aspect, R¹³ and R¹⁴ are each hydrogen whereinR_(X), R_(Y), and R_(X) are independently —CH₂CO₂R. In a further aspect,R is —CH₂OC(O)(CH₂)_(n)CH₃, wherein n is typically 0.

In an exemplary embodiment, modification of carboxylic groups withacetoxymethyl (AM) ester groups results in uncharged molecules than canpenetrate cell membranes—live cell versions of the heavy metal-bindingcompounds. This includes the carboxylic groups of the acetic acid groupsor other carboxylic groups on the metal chelating moiety. In thisparticular embodiment, typically at least one R is —CH₂OC(O)CH₃,preferably at least two R and most preferred at least three R areCH₂OC(O)CH₃. Once inside the cells, the lipophilic blocking groups arecleaved by nonspecific esterases revealing a metal chelating moiety ofthe present invention, e.g., a triacetic acid moiety. Alternatively,acetate groups on a compound of the present invention can also allow acompound to enter a live cell.

In another embodiment, R is H or a salt ion and the heavy metal-bindingcompounds of the present invention are used to bind and detect heavymetal ions that are not contained by a lipid bilayer. This includesheavy metal ions that are free in solution such as a biological fluid orheavy metal ions that have been released from cells and metal ionspresent in water samples, environmental samples, food stuff, etc. When Ris H or a salt ion the compounds are impermeant to cellular membranes.

The present heavy metal binding compound comprises two benzene ringsthat are joined by a C₁-C₃ hydrocarbon bridge (n is 1, 2 or 3)terminated by oxygen atoms, including methylenedioxy (—OCH₂O—),ethylenedioxy (—OCH₂CH₂O—) or propylenedioxy (—OCH₂CH₂CH₂O—) bridginggroups, where each benzene ring is optionally substituted by one or moresubstituents that adjust the metal ion-binding affinity, solubility,chemical reactivity, spectral properties or other physical properties ofthe compound. The benzene ring substituents (R¹, R², R³, R⁴, R⁵, R⁶, R⁷and R⁸) and the bridging group substituents (R⁹, R¹⁰, R¹¹ and R¹²) aretypically selected from substituents that are found on BAPTA compounds.This includes any substituents disclosed in U.S. Pat. Nos. 4,603,209;4,795,712; 4,849,362; 5,049,673; 5,453,517; 5,459,276; 5,516,911;5,501,980; 6,162,931 and 5,773,227.

In an exemplary embodiment, R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ areindependently selected from the group consisting of hydrogen, halogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, amino, substitutedamino, alkylamino, substituted alkylamino, aldehyde, carboxyl, azido,nitro, nitroso, cyano, thioether, sulfoalkyl, carboxyalkyl, aminoalkyl,reporter molecule, reactive group, carrier molecule or solid support.Each substituted amino substituent is independently substituted byhydrogen, C₁-C₆ alkyl, substituted alkyl, C₁-C₆ carboxyalkyl, analpha-acyloxyalkyl, a biologically compatible salt, aryl, substitutedaryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substitutedheteroaryl. Each alkyl portion is optionally substituted by halogen,amino, hydroxy, or amino.

Alternatively, any two adjacent substituents R¹-R⁸, taken incombination, form a fused ring moiety or reporter molecule. Specificallya member selected from R¹ in combination with R²; R² in combination withR³; R³ in combination with R⁴; R⁵ in combination with R⁶; R⁶ incombination with R⁷; and R⁷ in combination with R⁸; together with theatoms to which they are joined, form a ring which is a 5-, 6- or7-membered heterocycloalkyl, a substituted 5-, 6- or 7-memberedheterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, a substituted 5-,6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heteroaryl, asubstituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or 7-memberedaryl, a substituted 5-, 6- or 7-membered aryl or reporter molecule. Thering moieties may be independently substituted by halogen, azido, nitro,nitroso, amino, cyano, C₁-C₆ alkyl or C₁-C₆ alkoxy. Each alkyl portionis optionally substituted by halogen, amino, hydroxy, or amino.

In an exemplary embodiment, at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷and R⁸ is a carrier molecule, solid support, reactive group or reportermolecule. Typically, at least one of R³ or R⁶ is a carrier molecule,solid support, reactive group or reporter molecule.

The bridging group substituents, R⁹, R¹⁰, R¹¹ and R¹², are typicallyindependently selected from the group consisting of hydrogen, alkyl,carrier molecule, solid support, reactive group and reporter molecule.Alternatively, adjacent substituents R⁹ in combination with R¹⁰ or R¹¹in combination with R¹² together with the atoms to which they arejoined, form a ring which is a 5-, 6- or 7-membered heterocycloalkyl, asubstituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a5-, 6- or 7-membered heteroaryl, a substituted 5-, 6- or 7-memberedheteroaryl, a 5-, 6- or 7-membered aryl, or a substituted 5-, 6- or7-membered aryl.

In an exemplary embodiment each of R_(X), R_(Y), and R_(X)′ arehydrogen. In another embodiment at least one of R_(X), R_(Y), and R_(X)′is CH₂CO₂R. In one aspect R is hydrogen or a salt ion, in another aspectR is CH₂OC(O)CH₃.

In an exemplary embodiment, the present compounds further comprise areporter group to form a heavy metal indicator compound. These compoundsare particularly useful for detecting lead, cadmium and/or mercury ions.In one aspect the present compounds comprise exactly one reporter group,which include, but are not limited to, chromophore, fluorophore,fluorescent protein, phosphorescent dye or a tandem dye. In a furtheraspect, the reporter molecule is a chromophore or fluorophore that is axanthene, indole, cyanine, oxazole, dansyl, borapolyazaindacene,benzofuran, quinazolinone, benzazole, oxazine, pyrene, naphthalene,coumarin, biotin, enzyme substrate or fluorescent protein. Xanthenesinclude fluorescein or derivative thereof, rhodamine or derivativethereof, rhodol or derivative thereof or rosamine or a derivativethereof. These reporter molecules may also be substituted withsubstituents, including, but not limited to hydrogen, halogen, amino,substituted amino, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, sulfo, a lipophilic group(Am ester or acetate ester), solid support, reactive group or carriermolecule.

Thus, an exemplary embodiment, the present compounds further comprises acarrier molecule. Carrier molecules include, but are not limited to, anamino acid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid polymer, a hapten, apsoralen, a drug, a hormone, a lipid, a lipid assembly, a syntheticpolymer, a polymeric microparticle, a biological cell or a virus. In afurther aspect, the carrier molecule is an antibody or fragment thereof,an avidin or streptavidin, a biotin, a blood component protein, adextran, an enzyme, an enzyme inhibitor, a hormone, an IgG bindingprotein, a fluorescent protein, a growth factor, a lectin, alipopolysaccharide, a microorganism, a metal binding protein, a metalchelating moiety, a non-biological microparticle, a peptide toxin, aphosphotidylserine-binding protein, a structural protein, asmall-molecule drug, or a tyramide.

In yet another embodiment, the present compounds further comprise asolid support. Solid supports include, but are not limited to, amicrofluidic chip, a silicon chip, a microscope slide, a microplatewell, silica gels, polymeric membranes, particles, derivatized plasticfilms, glass beads, cotton, plastic beads, alumina gels,polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon,latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead.In one aspect, the solid support is sepharose, poly(acrylate),polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose,dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin,nitrocellulose, diazocellulose or starch

In an exemplary embodiment, the present compounds further comprise asolid support and a reporter group, which are particularly useful forhigh content screening.

In another embodiment, the present compounds comprise a reactive group.Reactive groups include, but are not limited to, an acrylamide, anactivated ester of a carboxylic acid, a carboxylic ester, an acyl azide,an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline,an amine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a reactive platinum complex, a silyl halide, a sulfonylhalide, a thiol or a photoactivatable group. In one aspect, the reactivegroup is carboxylic acid, succinimidyl ester of a carboxylic acid,hydrazide, amine or a maleimide.

In an exemplary embodiment, the present compounds are according to theformula:

with a biologically compatible anion wherein R is hydrogen, a salt ionor an AM ester represented by —CH₂OC(O)(CH₂)_(n)CH₃.

Synthesis

The general synthesis method involves joining a 2-nitrophenol derivativewith a catechol derivative via an ethylene bridge, followed by reductionof the nitro group to an amine to give representative compound A.Chelating moieties are then added to the aniline nitrogen and phenolicoxygen groups by reaction with electorophilic acetic acid esters, forexample to give representative compound B. Formylation of B affords C,which provides a reactive handle for attachment of fluorescent moieties.For example, in this case acid-mediated condensation of C with4-fluororesorcinol, followed by dehydrogenative oxidation, gives thefluorophore D in which the chelating acetic acid residues are stillprotected as esters. These esters can be cleaved by alkaline or acidichydrolysis to give metal ion-binding chelator E, in which thefluorescence is modulated by binding of certain transition metal orheavy metal ions. The carboxylates in E can be further derivatized intoother kinds of esters which facilitate the molecule's entry into livecells or hydrophobic environments. Properly chosen, these esters will bespontaneously hydrolyzed back to the free carboxylate form as in D, forexample trapping D in live cells so as to provide fluorescence reportingon the concentration of certain heavy metal or transition metal ions.

Additionally, compounds such as B can be nitrated and then the nitrogroup reduced so as to introduce an aniline moiety onto an aromaticring. This aniline moiety can be used as a reactive handle forattachment of fluorophores or other optical reporting agents such thatbinding of certain transition or heavy metal ions after cleavage of theacetic acid esters results in fluorescence changes.

Reporter Molecules

In an exemplary embodiment, the present compounds confer a detectablesignal, directly or indirectly, to the metal ions, wherein they arecovalently bonded to a reporter molecule. This results in the ability todetect, monitor and quantitate heavy metal ions in a sample.

Thus, in an exemplary embodiment, the present compound is covalentlybound to a reporter group, See Compound 10, 12 and 18. The reportermolecule can be attached to the compound through the chelating moiety bya linker or share atoms with the chelating moiety wherein no linker ispresent (See, Compound 20). If the compound has a reactive group, thenthe reporter molecule can alternatively be linked to the compoundthrough the reactive group. The reactive group may contain both areactive functional moiety and a linker, or only the reactive functionalmoiety.

The present reporter molecules can be any reporter molecule known to oneskilled in the art and when the reporter molecule is either covalentlylinked to a metal-chelating moiety or comprises part of themetal-chelating moiety wherein no linker is present, forms a metal ionbinding compound of the present invention that is useful for thedetection of lead, mercury and/or cadmium ions. Reporter moleculesinclude, without limitation, a dye, (chromophore or fluorophore), afluorescent protein, a phosphorescent dye, a tandem dye (energy transferpair), a microparticle, a hapten, an enzyme and a radioisotope.Preferred reporter molecules include dyes (both chromophores andfluorophores), fluorescent proteins, haptens, and enzymes. When thereporter molecule is a chromophore the heavy metal-binding compounds arechromogenic indicators, or more preferably, the reporter molecule is afluorophore, resulting in a compound that is a fluorogenic indicator forheavy metal ions. Therefore, binding a heavy metal ion to a heavymetal-binding compound results in a detectable optical response that canbe correlated to the presence of lead, cadmium and/or mercury ions.

In one embodiment, at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ isor is attached to a reporter molecule or any two adjacent R¹, R², R³,R⁴, R⁵, R⁶, R⁷, and R⁸ form a fused reporter molecule that share atomswith either ring of the present chelating moiety. In a particular aspectat least one of R², R³, R⁶, and R⁷, is or is attached to a reportermolecule. In a preferred aspect, either R³ or R⁶ is or is attached to areporter molecule.

Where the detectable response is a fluorescence response, it istypically a change in fluorescence, such as a change in the intensity,excitation or emission wavelength, distribution of fluorescence,fluorescence lifetime, fluorescence polarization, or a combinationthereof. Preferably, the detectable optical response upon binding atarget heavy metal ion is a change in fluorescence intensity that isgreater than approximately 10-fold relative to the same compound in theabsence of heavy metal ions, more preferably greater than 50-fold, andmost preferably more that 100-fold. This large increase in fluorescentsignal over baseline has not been previously observed with other heavymetal indicators that comprise a different metal chelating moiety. Otherwell known heavy metal indicators such as Phen Green (Invitrogen Corp.)indicate metal binding by fluorescence decreases. In another aspect, thedetectable optical response upon binding the target metal ion is a shiftin either the maximal excitation or emission wavelength or both that isgreater than about 20 nm, more preferably greater than about 30 nm.

A dye of the present invention is any chemical moiety that exhibits anabsorption maximum beyond 280 nm, and when covalently linked to a metalchelating moiety of the present invention, or shares atoms with themetal chelating moiety, forms a heavy metal-binding compound. Apreferred embodiment for detecting heavy metal ions in live cells orheavy metal ions secreted from live cells is a fluorogenic heavymetal-binding compound wherein the reporter molecule is dye. Asdescribed below in more detail, the covalent linkage can be a singlecovalent bond or a combination of stable chemical bonds. The covalentlinkage binding the dye to the metal chelating moiety is typically asingle covalent bond or a substituted alkyl chain that incorporates 1-20nonhydrogen atoms selected from the group consisting of C, N, O, S andP.

Dyes of the present invention include, without limitation; a pyrene, ananthracene, a naphthalene, an acridine, a stilbene, an indole orbenzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a carbocyanine (includingany corresponding compounds in U.S. Ser. Nos. 09/557,275; 09/968,401 and09/969,853 and U.S. Pat. Nos. 6,403,807; 6,348,599; 5,486,616;5,268,486; 5,569,587; 5,569,766; 5,627,027; 6,048,982 and 6,664,047), acarbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, aperylene, a pyridine, a quinoline, a borapolyazaindacene (including anycorresponding compounds disclosed in U.S. Pat. Nos. 4,774,339;5,187,288; 5,248,782; 5,274,113; and 5,433,896), a xanthene (includingany corresponding compounds disclosed in U.S. Pat. No. 6,162,931;6,130,101; 6,229,055; 6,339,392; 5,451,343 and U.S. Ser. No.09/922,333), an oxazine or a benzoxazine, a carbazine (including anycorresponding compounds disclosed in U.S. Pat. No. 4,810,636), aphenalenone, a coumarin (including an corresponding compounds disclosedin U.S. Pat. Nos. 5,696,157; 5,459,276; 5,501,980 and 5,830,912), abenzofuran (including an corresponding compounds disclosed in U.S. Pat.Nos. 4,603,209 and 4,849,362) and benzphenalenone (including anycorresponding compounds disclosed in U.S. Pat. No. 4,812,409) andderivatives thereof. As used herein, oxazines include resorufins(including any corresponding compounds disclosed in U.S. Pat. No.5,242,805), aminooxazinones, diaminooxazines, and theirbenzo-substituted analogs.

Where the dye is a xanthene, the dye is optionally a fluorescein, arhodol (including any corresponding compounds disclosed in U.S. Pat.Nos. 5,227,487 and 5,442,045), a rosamine or a rhodamine (including anycorresponding compounds in U.S. Pat. Nos. 5,798,276; 5,846,737;5,847,162; 6,017,712; 6,025,505; 6,080,852; 6,716,979; 6,562,632). Asused herein, fluorescein includes benzo- or dibenzofluoresceins,seminaphthofluoresceins, or naphthofluoresceins. Similarly, as usedherein rhodol includes seminaphthorhodafluors (including anycorresponding compounds disclosed in U.S. Pat. No. 4,945,171).

Preferred dyes of the invention include rhodol, fluorescein, rhodamine,dansyl, benzofuran, indole, cyanine, quinazolinone, pyrene, naphthalene,coumarin, oxazine, oxazole , benzofuran, indole, a benzazole andborapolyazaindacene. In one embodiment benzofuran and benzazole form afused reporter molecule with either benzo ring the chelating moiety. Inanother aspect, the reporter molecules xanthene, dansyl, benzofuran,indole, cyanine, quinazolinone, pyrene, naphthalene, coumarin, oxazine,indole, and borapolyazaindacene are independently attached to thechelating moiety by a linker.

In an exemplary embodiment, the dye contains one or more aromatic orheteroaromatic rings, that are optionally substituted one or more timesby a variety of substituents, including without limitation, hydrogen,amino, substituted amino, halogen, nitro, sulfo, cyano, alkyl,substituted alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl,arylalkyl, acyl, aryl or heteroaryl ring system, substituted aryl,benzo, solid support, reactive group, carrier molecule, lipophilicgroup, or other substituents typically present on chromophores orfluorophores known in the art.

In one aspect, the dyes are independently substituted by substituentsselected from the group consisting of hydrogen, halogen, amino,substituted amino, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, sulfo, reactive group, solidsupport, lipophilic group, and carrier molecule. In another embodiment,the xanthene dyes of this invention comprise both compounds substitutedand unsubstituted on the carbon atom of the central ring of the xantheneby substituents typically found in the xanthene-based dyes such asphenyl and substituted-phenyl moieties. Most preferred dyes arerhodamine, fluorescein, rhodal, rosamine and derivatives thereof. Thechoice of the dye attached to the chelating moiety will determine theheavy metal-binding compound's absorption and fluorescence emissionproperties.

In an exemplary embodiment, the dye has an absorption maximum beyond 480nm. In a particularly useful embodiment, the dye absorbs at or near 488nm to 514 nm (particularly suitable for excitation by the output of theargon-ion laser excitation source) or near 546 nm (particularly suitablefor excitation by a mercury arc lamp). As is the case for many dyes,they can also function as both chromophores and fluorophores, resultingin compounds that simultaneously act both as colorimetric andfluorescent labels for heavy metal ions. Thus, the described fluorescentdyes are also the preferred chromophores of the present invention.

For heavy metal-binding compounds that find use in detecting heavy metalions wherein a change in detectable signal is not required, i.e. unboundheavy metal-binding compounds can be washed away and the remaining heavymetal-binding compounds are bound to heavy metal ions, the alternativereporter molecules that are haptens, enzymes, fluorescent proteins andtandem dyes (energy transfer dyes) find use as reporter molecules of thepresent invention. In this aspect, a stable ternary complex is formedbetween a heavy metal ion and a heavy metal-binding molecule (e.g.,protein, carrier molecule or solid support). Therefore, these reportermolecules find use wherein the sample is immobilized on a solid orsemi-solid matrix or in biological fluids wherein a polarization assayis used to measure heavy metal ions or alternatively tandem dyes can beused resulting in a shift in signal when heavy metal ions are bound bythe heavy metal-binding compounds.

Enzymes are desirable reporter molecules because amplification of thedetectable signal can be obtained, resulting in increased assaysensitivity. The enzyme itself does not produce a detectable response,but functions to break down a substrate when it is contacted by anappropriate substrate such that the converted substrate produces afluorescent, colorimetric or luminescent signal. Enzymes amplify thedetectable signal because one enzyme on a heavy metal-binding compoundcan result in multiple substrate molecules being converted to adetectable signal. This is advantageous where there is a low quantity ofheavy metal ions present in the sample or a dye does not exist that willgive comparable or stronger signal than the enzyme. Dyes are mostpreferred because they do not require additional assay steps that canlead to an unstable heavy metal-binding complex and they do not lend tolive cell measurement of heavy metal ions. The enzyme substrate isselected to yield the preferred measurable product, e.g. color,fluorescence or chemiluminescence. Such substrates are extensively usedin the art, many of which are described in the MOLECULAR PROBESHANDBOOK, supra.

A preferred colorimetric or fluorogenic substrate and enzyme combinationuses oxidoreductases such as horseradish peroxidase (HRP) and asubstrate such as 3,3′-diaminobenzidine (DAB) or3-amino-9-ethylcarbazole (AEC), which yield a distinguishing color(brown and red, respectively). Other colorimetric oxidoreductasesubstrates that yield detectable products include, but are not limitedto: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPD), 3,3′,5,5′-tetramethylbenzidine (TMB),o-dianisidine, 5-aminosalicylic acid and 4-chloro-1-naphthol.Fluorogenic substrates include, but are not limited to, homovanillicacid or 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines andreduced benzothiazines, including Amplex® Red reagent and its variants(U.S. Pat. No. 4,384,042) and reduced dihydroxanthenes, includingdihydrofluoresceins (U.S. Pat. No. 6,162,931) and dihydrorhodamines,including dihydrorhodamine 123. Peroxidase substrates that are tyramides(U.S. Pat. Nos. 5,196,306; 5,583,001 and 5,731,158) represent a uniqueclass of peroxidase substrates in that they can be intrinsicallydetectable before action of the enzyme but are “fixed in place” by theaction of a peroxidase in the process described as tyramide signalamplification (TSA). These substrates are extensively utilized to labeltargets in samples that are cells, tissues or arrays for theirsubsequent detection by microscopy, flow cytometry, optical scanning andfluorometry.

Another preferred colorimetric (and in some cases fluorogenic) substrateand enzyme combination uses a phosphatase enzyme such as an acidphosphatase or a recombinant version of such a phosphatase incombination with a colorimetric substrate such as5-bromo-4-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolylphosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenylphosphate, or o-nitrophenyl phosphate or with a fluorogenic substratesuch as 4-methylumbelliferyl phosphate,6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, U.S. Pat.No. 5,830,912), fluorescein diphosphate, 3-O-methylfluoresceinphosphate, resorufin phosphate,9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAOphosphate), or ELF 97, ELF 39 or related phosphates (U.S. Pat. Nos.5,316,906 and 5,443,986).

Glycosidases, in particular β-galactosidase, β-glucuronidase andβ-glucosidase, are additional suitable enzymes. Appropriate colorimetricsubstrates include, but are not limited to, 5-bromo-4-chloro-3-indolylβ-D-galactopyranoside (X-gal) and similar indolyl galactosides,glucosides, and glucuronides, o-nitrophenyl β-D-galactopyranoside (ONPG)and p-nitrophenyl β-D-galactopyranoside. Preferred fluorogenicsubstrates include resorufin β-D-galactopyranoside, fluoresceindigalactoside (FDG), fluorescein diglucuronide and their structuralvariants (U.S. Pat. Nos. 5,208,148; 5,242,805; 5,362,628; 5,576,424 and5,773,236), 4-methylumbelliferyl β-D-galactopyranoside,carboxyumbelliferyl β-D-galactopyranoside and fluorinated coumarinβ-D-galactopyranosides (U.S. Pat. No. 5,830,912).

Additional enzymes include, but are not limited to, hydrolases such ascholinesterases and peptidases, oxidases such as glucose oxidase andcytochrome oxidases and reductases for which suitable substrates areknown.

Enzymes and their appropriate substrates that produce chemiluminescenceare preferred for some assays. These include, but are not limited to,natural and recombinant forms of luciferases and aequorins.Chemiluminescence-producing substrates for phosphatases, glycosidasesand oxidases such as those containing stable dioxetanes, luminol,isoluminol and acridinium esters are additionally useful. Severalchemiluminescent substrates for phosphatase enzymes are known, includingthe BOLD APB chemiluminescent substrate (Molecular Probes, Inc.).

In addition to enzymes, haptens such as biotin, digoxigenin and2,4-dinitrophenol are also preferred reporter molecules. Biotin isuseful because it can function in an enzyme system to further amplifythe detectable signal, and it can function as a tag to be used inaffinity chromatography for isolation purposes. For detection purposes,an enzyme conjugate that has affinity for biotin is used, such asavidin-HRP. Subsequently a peroxidase substrate is added to produce adetectable signal. For isolation purposes, a protein such as avidin thathas affinity for biotin is conjugated to agarose beads. The biotinlabeled metal-chelating moiety, after contacting a target heavy metalion, is then incubated with the avidin beads, on a column or insolution, to separate and/or concentrate the heavy metal ions. Apreferred form of biotin is the desthiobiotin analog, which can beeasily adsorbed and released from avidin-based affinity matrices. Apreferred form of avidin for some applications is CaptAvidinbiotin-binding protein (Molecular Probes), which permits facile releaseof biotinylated compounds.

Haptens also include, among other derivatives, hormones, naturallyoccurring and synthetic drugs, pollutants, allergens, affectormolecules, growth factors, chemokines, cytokines, lymphokines, aminoacids, peptides, chemical intermediates, nucleotides and the like.

Fluorescent proteins also find use as labels for the heavy metal-bindingcompounds of the present invention. Examples of fluorescent proteinsinclude green fluorescent protein (GFP) and the phycobiliproteins andthe derivatives thereof. The fluorescent proteins, especiallyphycobiliproteins, are particularly useful for creating tandemdye-reporter molecules or for indirect detection of hapten-labeled heavymetal-binding compounds or heavy metal-binding proteins that areimmobilized on a matrix, such as a microsphere or an array. These tandemdyes comprise a fluorescent protein and a fluorophore for the purposesof obtaining a larger Stokes shift, wherein the emission spectra arefarther shifted from the wavelength of the fluorescent protein'sabsorption spectra. This property is particularly advantageous fordetecting a low quantity of a target heavy metal ion in a sample whereinthe emitted fluorescent light is maximally optimized; in other words,little to none of the emitted light is reabsorbed by the fluorescentprotein. For this to work, the fluorescent protein and fluorophorefunction as an energy transfer pair wherein the fluorescent proteinemits at the wavelength that the acceptor fluorophore absorbs and thefluorophore then emits at a wavelength farther from the fluorescentproteins than could have been obtained with only the fluorescentprotein. Alternatively, the fluorophore functions as the energy donorand the fluorescent protein is the energy acceptor. Particularly usefulfluorescent proteins are the phycobiliproteins disclosed in U.S. Pat.Nos. 4,520,110; 4,859,582; 5,055,556 and the fluorophore bilin proteincombinations disclosed in U.S. Pat. No. 4,542,104. Alternatively, two ormore fluorophore dyes can function as an energy transfer pair whereinone fluorophore is a donor dye and the other is the acceptor dyeincluding any dye compounds disclosed in U.S. Pat. Nos. 6,358,684;5,863,727; 6,372,445; 6,221,606; 6,008,379; 5,945,526; 5,863,727;5,800,996; 6,335,440; 6,008,373; 6,184,379; 6,140,494 and 5,656,554.

Reactive Groups, Carrier Molecules and Solid Supports

The present compounds, in certain embodiments, are chemically reactivewherein the compounds comprise a reactive group. In a furtherembodiment, the compounds comprise a carrier molecule or solid support.These substituents, reactive groups, carrier molecules, and solidsupports, comprise a linker that is used to covalently attach thesubstituents to any of the moieties of the present compounds. The solidsupport, carrier molecule or reactive group may be directly attached(where linker is a single bond) to the moieties or attached through aseries of stable bonds, as disclosed above.

Any combination of linkers may be used to attach the carrier molecule,solid support or reactive group and the present compounds together. Thisdescription of the linker also applies to the reporter molecules, asdisclosed above. The linker may also be substituted to alter thephysical properties of the reporter moiety or chelating moiety, such asspectral properties of the dye. Examples of L include substituted orunsubstituted polyalkylene, arylene, alkylarylene, arylenealkyl, orarylthio moieties.

The linker typically incorporates 1-30 nonhydrogen atoms selected fromthe group consisting of C, N, O, S and P. The linker may be anycombination of stable chemical bonds, optionally including, single,double, triple or aromatic carbon-carbon bonds, as well ascarbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds,sulfur-sulfur bonds, carbon-sulfur bonds, phosphorus-oxygen bonds,phosphorus-nitrogen bonds, and nitrogen-platinum bonds. Typically thelinker incorporates less than 15 nonhydrogen atoms and are composed ofany combination of ether, thioether, thiourea, amine, ester,carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromaticbonds. Typically the linker is a combination of single carbon-carbonbonds and carboxamide, sulfonamide or thioether bonds. The bonds of thelinker typically result in the following moieties that can be found inthe linker: ether, thioether, carboxamide, thiourea, sulfonamide, urea,urethane, hydrazine, alkyl, aryl, heteroaryl, alkoxy, cycloalkyl andamine moieties. Examples of a linker include substituted orunsubstituted polymethylene, arylene, alkylarylene, arylenealkyl, andarylthio.

In one embodiment, the linker contains 1-6 carbon atoms; in another, thelinker comprises a thioether linkage. Exemplary linking members includea moiety that includes —C(O)NH—, —C(O)O—, —NH—, —S—, —O—, and the like.In another embodiment, the linker is or incorporates the formula—(CH₂)_(d)(CONH(CH₂)_(e))_(z)— or where d is an integer from 0-5, e isan integer from 1-5 and z is 0 or 1. In a further embodiment, the linkeris or incorporates the formula —O—(CH₂)—. In yet another embodiment, thelinker is or incorporates a phenylene or a 2-carboxy-substitutedphenylene.

An important feature of the linker is to provide an adequate spacebetween the carrier molecule, reactive group or solid support and thedye so as to prevent steric hinderance. Therefore, the linker of thepresent compound is important for (1) attaching the carrier molecule,reactive group or solid support to the compound, (2) providing anadequate space between the carrier molecule, reactive group or solidsupport and the compound so as not to sterically hinder the action ofthe compound and (3) for altering the physical properties of the presentcompounds.

In another exemplary embodiment of the invention, the present compoundsare chemically reactive, and are substituted by at least one reactivegroup. The reactive group functions as the site of attachment foranother moiety, such as a carrier molecule or a solid support, whereinthe reactive group chemically reacts with an appropriate reactive orfunctional group on the carrier molecule or solid support.

Reactive groups or reactive group precursors may be positioned duringthe formation of the present compounds. Thus, compounds incorporating areactive group can be reacted with and attached to a wide variety ofbiomolecules or non-biomolecules that contain or are modified to containfunctional groups with suitable reactivity. When a labeled componentincludes a compound as disclosed herein, then this conjugate typicallypossesses the nucleic acid staining abilities of the parent compound,particularly DNA staining. However, the present fluorescent compoundscan also function as reporter molecules for the labeled componentswherein the nucleic acid binding properties of the reagents may notemployed.

Preferred reactive groups for incorporation into the disclosed compoundsmay be selected to react with an amine, a thiol or an alcohol. In anexemplary embodiment, the compounds of the invention further comprise areactive group that is an acrylamide, an activated ester of a carboxylicacid, a carboxylic ester, an acyl azide, an acyl nitrile, an aldehyde,an alkyl halide, an anhydride, an aniline, an amine, an aryl halide, anazide, an aziridine, a boronate, a diazoalkane, a haloacetamide, ahaloalkyl, a halotriazine, a hydrazine, an imido ester, an isocyanate,an isothiocyanate, a maleimide, a phosphoramidite, a photoactivatablegroup, a reactive platinum complex, a silyl halide, a sulfonyl halide,and a thiol. In a particular embodiment the reactive group is selectedfrom the group consisting of carboxylic acid, succinimidyl ester of acarboxylic acid, hydrazide, amine and a maleimide. In exemplaryembodiment, at least one member selected from R¹, R², R³, R⁴, R⁵, R⁶,R⁷, R⁸, R⁹, R¹⁰, R¹¹ or R¹² comprises a reactive group. Preferably, atleast one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸ comprises a reactivegroup or is attached to a reactive group. More preferred, at least oneof R³ or R⁶ comprises a reactive group or is attached to a reactivegroup. Alternatively, if the present compound comprises a carriermolecule or solid support a reactive group may be covalently attachedindependently to those substituents, allowing for further conjugation toa another dye, carrier molecule or solid support.

In one aspect, the compound comprises at least one reactive group thatselectively reacts with an amine group. This amine-reactive group isselected from the group consisting of succinimidyl ester, sulfonylhalide, tetrafluorophenyl ester and iosothiocyanates. Thus, in oneaspect, the present compounds form a covalent bond with anamine-containing molecule in a sample. In another aspect, the compoundcomprises at least one reactive group that selectively reacts with athiol group. This thiol-reactive group is selected from the groupconsisting of maleimide, haloalkyl and haloacetamide (including anyreactive groups disclosed in U.S. Pat. Nos. 5,362,628; 5,352,803 and5,573,904).

The pro-reactive groups are synthesized during the formation of themonomer moieties and carrier molecule and solid support containingcompounds to provide chemically reactive compounds. In this way,compounds incorporating a reactive group can be covalently attached to awide variety of carrier molecules or solid supports that contain or aremodified to contain functional groups with suitable reactivity,resulting in chemical attachment of the components. In an exemplaryembodiment, the reactive group of the compounds of the invention and thefunctional group of the carrier molecule or solid support compriseelectrophiles and nucleophiles that can generate a covalent linkagebetween them. Alternatively, the reactive group comprises aphotoactivatable group, which becomes chemically reactive only afterillumination with light of an appropriate wavelength. Typically, theconjugation reaction between the reactive group and the carrier moleculeor solid support results in one or more atoms of the reactive groupbeing incorporated into a new linkage attaching the present compound ofthe invention to the carrier molecule or solid support. Selectedexamples of functional groups and linkages are shown in Table 1, wherethe reaction of an electrophilic group and a nucleophilic group yields acovalent linkage.

TABLE 1 Examples of some routes to useful covalent linkages ResultingCovalent Electrophilic Group Nucleophilic Group Linkage activatedesters* amines/anilines carboxamides acrylamides thiols thioethers acylazides** amines/anilines carboxamides acyl halides amines/anilinescarboxamides acyl halides alcohols/phenols esters acyl nitrilesalcohols/phenols esters acyl nitriles amines/anilines carboxamidesaldehydes amines/anilines imines aldehydes or ketones hydrazineshydrazones aldehydes or ketones hydroxylamines oximes alkyl halidesamines/anilines alkyl amines alkyl halides carboxylic acids esters alkylhalides thiols thioethers alkyl halides alcohols/phenols ethers alkylsulfonates thiols thioethers alkyl sulfonates carboxylic acids estersalkyl sulfonates alcohols/phenols ethers anhydrides alcohols/phenolsesters anhydrides amines/anilines carboxamides aryl halides thiolsthiophenols aryl halides amines aryl amines aziridines thiols thioethersboronates glycols boronate esters carbodiimides carboxylic acidsN-acylureas or anhydrides diazoalkanes carboxylic acids esters epoxidesthiols thioethers haloacetamides thiols thioethers haloplatinate aminoplatinum complex haloplatinate heterocycle platinum complexhaloplatinate thiol platinum complex halotriazines amines/anilinesaminotriazines halotriazines alcohols/phenols triazinyl ethershalotriazines thiols triazinyl thioethers imido esters amines/anilinesamidines isocyanates amines/anilines ureas isocyanates alcohols/phenolsurethanes isothiocyanates amines/anilines thioureas maleimides thiolsthioethers phosphoramidites alcohols phosphite esters silyl halidesalcohols silyl ethers sulfonate esters amines/anilines alkyl aminessulfonate esters thiols thioethers sulfonate esters carboxylic acidsesters sulfonate esters alcohols ethers sulfonyl halides amines/anilinessulfonamides sulfonyl halides phenols/alcohols sulfonate esters*Activated esters, as understood in the art, generally have the formula—COΩ, where Ω is a good leaving group (e.g., succinimidyloxy (—OC₄H₄O₂)sulfosuccinimidyloxy (—OC₄H₃O₂—SO₃H), -1-oxybenzotriazolyl (—OC₆H₄N₃);or an aryloxy group or aryloxy substituted one or more times by electronwithdrawing substituents such as nitro, fluoro, chloro, cyano, ortrifluoromethyl, or combinations thereof, used to form activated arylesters; or a carboxylic acid activated by a carbodiimide to form ananhydride or mixed anhydride —OCOR^(a) or —OCNR^(a)NHR^(b), where R^(a)and R^(b), which may be the same or different, are C₁-C₆ alkyl, C₁-C₆perfluoroalkyl, or C₁-C₆ alkoxy; or cyclohexyl, 3-dimethylaminopropyl,or N-morpholinoethyl). **Acyl azides can also rearrange to isocyanates

Choice of the reactive group used to attach the compound of theinvention to the substance to be conjugated typically depends on thereactive or functional group on the substance to be conjugated and thetype or length of covalent linkage desired. The types of functionalgroups typically present on the organic or inorganic substances(biomolecule or non-biomolecule) include, but are not limited to,amines, amides, thiols, alcohols, phenols, aldehydes, ketones,phosphates, imidazoles, hydrazines, hydroxylamines, disubstitutedamines, halides, epoxides, silyl halides, carboxylate esters, sulfonateesters, purines, pyrimidines, carboxylic acids, olefinic bonds, or acombination of these groups. A single type of reactive site may beavailable on the substance (typical for polysaccharides or silica), or avariety of sites may occur (e.g., amines, thiols, alcohols, phenols), asis typical for proteins.

Typically, the reactive group will react with an amine, a thiol, analcohol, an aldehyde, a ketone, or with silica. Preferably, reactivegroups react with an amine or a thiol functional group, or with silica.In one embodiment, the reactive group is an acrylamide, an activatedester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde,an alkyl halide, a silyl halide, an anhydride, an aniline, an arylhalide, an azide, an aziridine, a boronate, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazine (including hydrazides), animido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a reactive platinum complex, a sulfonyl halide, or athiol group. By “reactive platinum complex” is particularly meantchemically reactive platinum complexes such as described in U.S. Pat.No. 5,714,327.

Where the reactive group is an activated ester of a carboxylic acid,such as a succinimidyl ester of a carboxylic acid, a sulfonyl halide, atetrafluorophenyl ester or an isothiocyanates, the resulting compound isparticularly useful for preparing conjugates of carrier molecules suchas proteins, nucleotides, oligonucleotides, or haptens. Where thereactive group is a maleimide, haloalkyl or haloacetamide (including anyreactive groups disclosed in U.S. Pat. Nos. 5,362,628; 5,352,803 and5,573,904 (supra)) the resulting compound is particularly useful forconjugation to thiol-containing substances. Where the reactive group isa hydrazide, the resulting compound is particularly useful forconjugation to periodate-oxidized carbohydrates and glycoproteins, andin addition is an aldehyde-fixable polar tracer for cell microinjection.Where the reactive group is a silyl halide, the resulting compound isparticularly useful for conjugation to silica surfaces, particularlywhere the silica surface is incorporated into a fiber optic probesubsequently used for remote ion detection or quantitation.

In a particular aspect, the reactive group is a photoactivatable groupsuch that the group is only converted to a reactive species afterillumination with an appropriate wavelength. An appropriate wavelengthis generally a UV wavelength that is less than 400 nm. This methodprovides for specific attachment to only the target molecules, either insolution or immobilized on a solid or semi-solid matrix.Photoactivatable reactive groups include, without limitation,benzophenones, aryl azides and diazirines.

Preferably, the reactive group is a photoactivatable group, succinimidylester of a carboxylic acid, a haloacetamide, haloalkyl, a hydrazine, anisothiocyanate, a maleimide group, an aliphatic amine, a silyl halide, acadaverine or a psoralen. More preferably, the reactive group is asuccinimidyl ester of a carboxylic acid, a maleimide, an iodoacetamide,or a silyl halide. In a particular embodiment the reactive group is asuccinimidyl ester of a carboxylic acid, a sulfonyl halide, atetrafluorophenyl ester, an iosothiocyanates or a maleimide.

The selection of a covalent linkage to attach the reporter molecule tothe carrier molecule or solid support typically depends on thechemically reactive group on the component to be conjugated. Thediscussion regarding reactive groups in the section immediatelypreceding is relevant here as well. Exemplary reactive groups typicallypresent on the biological or non-biological components include, but arenot limited to, amines, thiols, alcohols, phenols, aldehydes, ketones,phosphates, imidazoles, hydrazines, hydroxylamines, disubstitutedamines, halides, epoxides, sulfonate esters, purines, pyrimidines,carboxylic acids, or a combination of these groups. A single type ofreactive site may be available on the component (typical forpolysaccharides), or a variety of sites may occur (e.g. amines, thiols,alcohols, phenols), as is typical for proteins. A carrier molecule orsolid support may be conjugated to more than one reporter molecule,which may be the same or different, or to a substance that isadditionally modified by a hapten. Although some selectivity can beobtained by careful control of the reaction conditions, selectivity oflabeling is best obtained by selection of an appropriate reactivecompound.

In another exemplary embodiment, the present compound is covalentlybound to a carrier molecule. If the compound has a reactive group, thenthe carrier molecule can alternatively be linked to the compound throughthe reactive group. The reactive group may contain both a reactivefunctional moiety and a linker, or only the reactive functional moiety.

A variety of carrier molecules are useful in the present invention.Exemplary carrier molecules include antigens, steroids, vitamins, drugs,haptens, metabolites, toxins, environmental pollutants, amino acids,peptides, proteins, nucleic acids, nucleic acid polymers, carbohydrates,lipids, and polymers. In exemplary embodiment, at least one memberselected from R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ or R¹²comprises a carrier molecule. Preferably, at least one of R¹, R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, comprises a carrier molecule or is attached to acarrier molecule. More preferably, at least one of R³ or R⁶ comprises acarrier molecule or is attached to a carrier molecule. Alternatively, ifthe present compound comprises a reactive group or solid support areactive group may be covalently attached independently to thosesubstituents, allowing for further conjugation to a reactive group,carrier molecule or solid support.

In an exemplary embodiment, the carrier molecule comprises an aminoacid, a peptide, a protein, a polysaccharide, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, adrug, a hormone, a lipid, a lipid assembly, a synthetic polymer, apolymeric microparticle, a biological cell, a virus and combinationsthereof. In another exemplary embodiment, the carrier molecule isselected from a hapten, a nucleotide, an oligonucleotide, a nucleic acidpolymer, a protein, a peptide or a polysaccharide. In a preferredembodiment the carrier molecule is amino acid, a peptide, a protein, apolysaccharide, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipidassembly, a tyramine, a synthetic polymer, a polymeric microparticle, abiological cell, cellular components, an ion chelating moiety, anenzymatic substrate or a virus. In another preferred embodiment, thecarrier molecule is an antibody or fragment thereof, an antigen, anavidin or streptavidin, a biotin, a dextran, an antibody bindingprotein, a fluorescent protein, agarose, and a non-biologicalmicroparticle. Typically, the carrier molecule is an antibody, anantibody fragment, antibody-binding proteins, avidin, streptavidin, atoxin, a lectin, or a growth factor. Preferred haptens include biotin,digoxigenin and fluorophores.

Antibody binging proteins include, but are not limited to, protein A,protein G, soluble Fc receptor, protein L, lectins, anti-IgG, anti-IgA,anti-IgM, anti-IgD, anti-IgE or a fragment thereof.

In an exemplary embodiment, the enzymatic substrate is selected from anamino acid, peptide, sugar, alcohol, alkanoic acid, 4-guanidinobenzoicacid, nucleic acid, lipid, sulfate, phosphate, —CH₂OCOalkyl andcombinations thereof. Thus, the enzyme substrates can be cleave byenzymes selected from the group consisting of peptidase, phosphatase,glycosidase, dealkylase, esterase, guanidinobenzotase, sulfatase,lipase, peroxidase, histone deacetylase, endoglycoceramidase,exonuclease, reductase and endonuclease.

In another exemplary embodiment, the carrier molecule is an amino acid(including those that are protected or are substituted by phosphates,carbohydrates, or C₁ to C₂₂ carboxylic acids), or a polymer of aminoacids such as a peptide or protein. In a related embodiment, the carriermolecule contains at least five amino acids, more preferably 5 to 36amino acids. Exemplary peptides include, but are not limited to,neuropeptides, cytokines, toxins, protease substrates, and proteinkinase substrates. Other exemplary peptides may function as organellelocalization peptides, that is, peptides that serve to target theconjugated compound for localization within a particular cellularsubstructure by cellular transport mechanisms. Preferred protein carriermolecules include enzymes, antibodies, lectins, glycoproteins, histones,albumins, lipoproteins, avidin, streptavidin, protein A, protein G,phycobiliproteins and other fluorescent proteins, hormones, toxins andgrowth factors. Typically, the protein carrier molecule is an antibody,an antibody fragment, avidin, streptavidin, a toxin, a lectin, or agrowth factor. Exemplary haptens include biotin, digoxigenin andfluorophores.

In another exemplary embodiment, the carrier molecule comprises anucleic acid base, nucleoside, nucleotide or a nucleic acid polymer,optionally containing an additional linker or spacer for attachment of afluorophore or other ligand, such as an alkynyl linkage (U.S. Pat. No.5,047,519), an aminoallyl linkage (U.S. Pat. No. 4,711,955) or otherlinkage. In another exemplary embodiment, the nucleotide carriermolecule is a nucleoside or a deoxynucleoside or a dideoxynucleoside.

Exemplary nucleic acid polymer carrier molecules are single- ormulti-stranded, natural or synthetic DNA or RNA oligonucleotides, orDNA/RNA hybrids, or incorporating an unusual linker such as morpholinederivatized phosphates (AntiVirals, Inc., Corvallis Oreg.), or peptidenucleic acids such as N-(2-aminoethyl)glycine units, where the nucleicacid contains fewer than 50 nucleotides, more typically fewer than 25nucleotides.

In another exemplary embodiment, the carrier molecule comprises acarbohydrate or polyol that is typically a polysaccharide, such asdextran, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, starch,agarose and cellulose, or is a polymer such as a poly(ethylene glycol).In a related embodiment, the polysaccharide carrier molecule includesdextran, agarose or FICOLL.

In another exemplary embodiment, the carrier molecule comprises a lipid(typically having 6-25 carbons), including glycolipids, phospholipids,and sphingolipids. Alternatively, the carrier molecule comprises a lipidvesicle, such as a liposome, or is a lipoprotein (see below). Somelipophilic substituents are useful for facilitating transport of theconjugated dye into cells or cellular organelles.

Alternatively, the carrier molecule is cells, cellular systems, cellularfragments, or subcellular particles. Examples of this type of conjugatedmaterial include virus particles, bacterial particles, virus components,biological cells (such as animal cells, plant cells, bacteria, oryeast), or cellular components. Examples of cellular components that canbe labeled, or whose constituent molecules can be labeled, include butare not limited to lysosomes, endosomes, cytoplasm, nuclei, histones,mitochondria, Golgi apparatus, endoplasmic reticulum and vacuoles.

In an exemplary embodiment, the carrier molecule comprises a specificbinding pair member wherein the present compounds are conjugated to aspecific binding pair member and are used to detect a heavy metal ion inclose proximity to the complimentary member of the specific bindingpair. Exemplary binding pairs are set forth in Table 2.

TABLE 2 Representative Specific Binding Pairs antigen antibody biotinavidin (or streptavidin or anti- biotin) IgG* protein A or protein Gdrug drug receptor folate folate binding protein toxin toxin receptorcarbohydrate lectin or carbohydrate receptor peptide peptide receptorprotein protein receptor enzyme substrate enzyme DNA (RNA) cDNA (cRNA)†hormone hormone receptor ion chelator antibody antibody-binding proteins*IgG is an immunoglobulin †cDNA and cRNA are the complementary strandsused for hybridization

In an exemplary embodiment, the present compounds of the invention arecovalently bonded to a solid support. The solid support may be attachedto the compound or through a reactive group, if present, or through acarrier molecule, if present. In exemplary embodiment, at least onemember selected from R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ andR¹² comprises a solid support. Preferably, at least one of R¹, R², R³,R⁴, R⁵, R⁶, R⁷, or R⁸ comprises a solid support or is attached to asolid support. More preferred, at least one of R³ or R⁶ comprises asolid support or is attached to a solid support. Alternatively, if thepresent compound comprises a carrier molecule or reactive group a solidsupport may be covalently attached independently to those substituents,allowing for further conjugation to a another dye, carrier molecule orsolid support.

A solid support suitable for use in the present invention is typicallysubstantially insoluble in liquid phases. Solid supports of the currentinvention are not limited to a specific type of support. Rather, a largenumber of supports are available and are known to one of ordinary skillin the art. Thus, useful solid supports include solid and semi-solidmatrixes, such as aerogels and hydrogels, resins, beads, biochips(including thin film coated biochips), microfluidic chip, a siliconchip, multi-well plates (also referred to as microtitre plates ormicroplates), membranes, conducting and nonconducting metals, glass(including microscope slides) and magnetic supports. More specificexamples of useful solid supports include silica gels, polymericmembranes, particles, derivatized plastic films, glass beads, cotton,plastic beads, alumina gels, polysaccharides such as Sepharose,poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar,cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin,mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride,polypropylene, polyethylene (including poly(ethylene glycol)), nylon,latex bead, magnetic bead, paramagnetic bead, superparamagnetic bead,starch and the like.

In some embodiments, the solid support may include a solid supportreactive functional group, including, but not limited to, hydroxyl,carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea,carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide,sulfoxide, etc., for attaching the compounds of the invention. Usefulreactive groups are disclosed above and are equally applicable to thesolid support reactive functional groups herein.

A suitable solid phase support can be selected on the basis of desiredend use and suitability for various synthetic protocols. For example,where amide bond formation is desirable to attach the compounds of theinvention to the solid support, resins generally useful in peptidesynthesis may be employed, such as polystyrene (e.g., PAM-resin obtainedfrom Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE™ resin(obtained from Aminotech, Canada), polyamide resin (obtained fromPeninsula Laboratories), polystyrene resin grafted with polyethyleneglycol (TentaGel™, Rapp Polymere, Tubingen, Germany),polydimethyl-acrylamide resin (available from Milligen/Biosearch,California), or PEGA beads (obtained from Polymer Laboratories).

While it has been stressed that a wide range of components can be usedto make the heavy metal-binding compounds it should also be understoodthat the individual selection of components to make a particularlyuseful heavy metal-binding compound for detection purposes requires anunderstanding of the reporter molecules, carrier molecules, reactivegroup, solid supports, the linkers, the metal chelating moiety and howcertain combinations, and substituents function to selectively bind tophysiological concentrations of heavy metal ions in the presence ofphysiological concentrations of calcium ions or other non-target metalions.

Preparation of Conjugates

Conjugates of components (carrier molecules or solid supports), e.g.,drugs, peptides, toxins, nucleotides, phospholipids and other organicmolecules are prepared by organic synthesis methods using the reactivedyes, are generally prepared by means well recognized in the art(Haugland, MOLECULAR PROBES HANDBOOK, supra, 2002). Conjugation to forma covalent bond may consist of simply mixing the reactive compounds ofthe present invention in a suitable solvent in which both the reactivecompound and the substance to be conjugated are soluble. The reactionpreferably proceeds spontaneously without added reagents at roomtemperature or below. For those reactive dyes that are photoactivated,conjugation is facilitated by illumination of the reaction mixture toactivate the reactive dye. Chemical modification of water-insolublesubstances, so that a desired dye-conjugate may be prepared, ispreferably performed in an aprotic solvent such as dimethylformamide(DMF), dimethylsulfoxide (DMSO), acetone, ethyl acetate, toluene, orchloroform.

Preparation of peptide or protein conjugates typically comprises firstdissolving the protein to be conjugated in aqueous buffer at about.1-10mg/mL at room temperature or below. Bicarbonate buffers (pH about 8.3)are especially suitable for reaction with succinimidyl esters, phosphatebuffers (pH about 7.2-8) for reaction with thiol-reactive functionalgroups and carbonate or borate buffers (pH about 9) for reaction withisothiocyanates and dichlorotriazines. The appropriate reactive compoundis then dissolved in a nonhydroxylic solvent (usually DMSO or DMF) in anamount sufficient to give a suitable degree of conjugation when added toa solution of the protein to be conjugated. The appropriate amount ofcompound for any protein or other component is convenientlypredetermined by experimentation in which variable amounts of thepresent compound are added to the protein, the conjugate ischromatographically purified to separate unconjugated compound and thecompound-protein conjugate is tested in its desired application.

Following addition of the reactive compound to the component solution,the mixture may be incubated for a suitable period (typically about 1hour at room temperature to several hours on ice), the excess unreactedcompound is removed by gel filtration, dialysis, HPLC, adsorption on anion exchange or hydrophobic polymer or other suitable means. Theconjugate is used in solution or lyophilized. In this way, suitableconjugates can be prepared from antibodies, antibody fragments, avidins,lectins, enzymes, proteins A and G, cellular proteins, albumins,histones, growth factors, hormones, and other proteins. The approximatedegree of substitution is determined from the long wavelength absorptionof the compound-protein conjugate by using the extinction coefficient ofthe un-reacted compound at its long wavelength absorption peak, theunmodified protein's absorption peak in the ultraviolet and bycorrecting the UV absorption of the conjugate for absorption by thecompound in the UV.

Conjugates of polymers, including biopolymers and other higher molecularweight polymers are typically prepared by means well recognized in theart (for example, Brinkley et al., Bioconjugate Chem., 3: 2 (1992)). Inthese embodiments, a single type of reactive site may be available, asis typical for polysaccharides or multiple types of reactive sites (e.g.amines, thiols, alcohols, phenols) may be available, as is typical forproteins. Selectivity of labeling is best obtained by selection of anappropriate reactive compound. For example, modification of thiols witha thiol-selective reagent such as a haloacetamide or maleimide, ormodification of amines with an amine-reactive reagent such as anactivated ester, acyl azide, isothiocyanate or3,5-dichloro-2,4,6-triazine. Partial selectivity can also be obtained bycareful control of the reaction conditions.

When modifying polymers with the compounds, an excess of the compound istypically used, relative to the expected degree of compoundsubstitution. Any residual, un-reacted compound or hydrolysis product istypically removed by dialysis, chromatography or precipitation. Presenceof residual, unconjugated compound can be detected by thin layerchromatography using a solvent that elutes the compound away from itsconjugate. In all cases it is usually preferred that the reagents bekept as concentrated as practical so as to obtain adequate rates ofconjugation.

In an exemplary embodiment, the conjugate is associated with anadditional substance that binds either to the compound or the component(reporter molecule, carrier molecule, solid support) through noncovalentinteraction. In another exemplary embodiment, the additional substanceis an antibody, an enzyme, a hapten, a lectin, a receptor, anoligonucleotide, a nucleic acid, a liposome, or a polymer. Theadditional substance is optionally used to probe for the location of theconjugate, for example, as a means of enhancing the signal of theconjugate.

Methods of Use

The heavy metal-binding compounds of the present invention are usefulfor any application where it is desirable to complex a target metal ion(lead, cadmium, mercury, nickel and/or lanthanum). Thus, the presentcompounds may be utilized without limit for the detection, monitoring,quantitation, binding and isolating of heavy metal ions. Selected heavymetal-binding compounds of the invention may be useful as ionophores,that is, they facilitate the transport of selected target ions acrosscell membranes. Where the heavy metal-binding compound is bound to acarrier molecule or solid support that is a polymeric matrix, such as amicroparticle, dextran, polystyrene or agarose, the compounds are usefulfor depleting a sample solution, sequestering, of a selected target ion,particularly where the polymeric matrix is used to pack a chromatographycolumn. Other heavy metal-binding compounds (those bound to a reportermolecule) are useful as fluorescent, colorimetric or fluorometricindicators for a selected target ion. This new class of heavymetal-binding compounds can be used in any of the same assays previouslydescribed for heavy metal indicators and other physiological metal ionindicators.

In an exemplary embodiment, the present methods comprise detecting thepresence or absence of a metal ion in a sample. The steps of the methodcomprises combining a present metal ion reporter molecule with thesample to prepare a labeling mixture; incubating the labeling mixturefor a sufficient amount of time for the metal ion reporter molecule toassociate with the metal ion in the sample to form an incubated mixture;illuminating the incubated sample with an appropriate wavelength to forman illuminated mixture; and, observing the illuminated mixture wherebythe presence or absence of the metal ion in a sample is detected.

The compound is typically combined with the sample as a stainingsolution. The staining solution is typically prepared by dissolving apresent metal ion reporter molecule in an aqueous solvent such as water,a buffer solution or assay solution, such as phosphate buffered saline,or an organic solvent such as dimethylsulfoxide (DMSO),dimethylformamide (DMF), methanol, ethanol or acetonitrile. Typically,the present metal ion reporter molecules are first dissolved in anorganic solvent such as DMSO as a stock solution. The stock solution istypically prepared about 300-10× more concentrated that the effectiveworking concentration. Thus, the stock solution is diluted to aneffective working concentration in an aqueous solution that optionallyincludes appropriate buffering components. Aqueous solution differdepending on the assay format. For detection of heavy metal ions in livecells an appropriate buffer is normal (physiological) saline, PBS(phosphate buffered saline) Hanks media, RPMI media or other cellculture media. For detection of heavy metal ions in a solution basedassay format appropriate aqueous dilution solution include MOPS, Good'sbuffer, and PIPES buffers. Additional buffering components include suchas, 50-100 mM formate buffer, pH 4.0, sodium citrate, pH 4.5, sodiumacetate, pH 5.0, MES, pH 6.0, imidazole, pH 7.0, HEPES, pH 6.8, Trisacetate, pH 8.0, Tris-HCl, pH 8.5, Tris borate, pH 9.0 and sodiumbicarbonate, pH 10.

An effective working concentration of the present compounds is theamount sufficient to give a detectable optical response when complexedwith heavy metal ions. Typically, the effective amount is about 10 nM to100 μM. Most preferred is about 200 nM to 5 μM. For selected reportercompounds, staining is optimal when the staining solution has aconcentration of about 400 nm to about 1 μM (see Examples 8-16). It isgenerally understood that the specific amount of the metal ion reportermolecules present in a staining solution is determined by the physicalnature of the sample and the nature of the analysis being performed.

Initially, the suitability of a heavy metal-binding compound as anindicator of heavy metal ion concentration is commonly tested by mixinga constant amount of the indicator with a measured amount of the targetion under the expected experimental conditions.

In general, this colorimetric or fluorometric method comprises combiningheavy metal-binding compounds of the present invention with a sample fora sufficient time to allow said compounds to bind heavy metal ionswhereby heavy metal ions are bound. Following binding of the presentheavy metal ions, the sample is illuminated with an appropriate lightsource and the signal correlated with the known concentration of heavymetal ions. This titration curve is then used to experimentallydetermine the appropriate heavy metal-binding compound for a particularassay.

Preferred indicators display a high selectivity, that is, they show asufficient rejection of non-target ions. The interference of anon-target ion is tested by a comparable titration of the indicator withthat ion. Although preferred target ions for most indicators of thepresent invention are lead, cadmium, nickel, lanthanum, and mercury, anyion that yields a detectable change in absorption wavelengths, emissionwavelengths, fluorescence lifetimes or other measurable optical propertyover the concentration range of interest is potentially measured usingone of the indicators of this invention.

The sample may be combined with the staining solution by any means thatfacilitates contact between the metal ion reporter molecules and themetal ions. The contact can occur through simple mixing, as in the casewhere the sample is a solution. The present reporter molecules may beadded to the sample directly or may contact the sample on an inertmatrix such as a blot or gel, a testing strip, a microarray, or anyother solid or semi-solid surface, for example where only a simple andvisible demonstration of the presence of metal ions is desired. Anyinert matrix used to separate the sample can be used to detect thepresence of nucleic acids by observing the fluorescent response on theinert matrix. Thus, in one embodiment is provided a compositioncomprising a sample and a present metal ion reporter molecule.

Alternatively, the sample may include cells and/or cell membranes. Whileselected examples of the compound disclosed herein may permeate cellularmembranes rapidly and completely upon addition of the staining solution,such as those comprising lipophilic moieties, any technique that issuitable for transporting the reporter molecules across cell membraneswith minimal disruption of the viability of the cell and integrity ofcell membranes is a valid method of combining the sample with thepresent reporter molecules for detection of intracellular metal ions.Examples of suitable processes include action of chemical agents such asdetergents, enzymes or adenosine triphosphate; receptor- or transportprotein-mediated uptake; pore-forming proteins; microinjection;electroporation; hypoosmotic shock; or minimal physical disruption suchas scrape loading or bombardment with solid particles coated with or inthe presence of the present reporter molecules.

The sample is incubated in the presence of the metal ion reportermolecules for a time sufficient to form the fluorogenic metalion-reporter molecule complex. Detectable fluorescence in a solution ofmetal ions is essentially instantaneous. Detectable fluorescence withincell membranes requires the permeation of the dye into the cell. Ingeneral, visibly detectable fluorescence can be obtained in a widevariety of cells with certain cell permeant embodiments of the presentinvention within about 10-30 minutes after combination with the sample,commonly within about 10-20 minutes. While permeation and fluorescenceshould be rapid for all reporter molecules comprising a lipophilicmoiety such as an AM ester, it is readily apparent to one skilled in theart that the time necessary for sufficient permeation of the dye, orsufficient formation of the fluorescent metal ion complex, is dependentupon the physical and chemical nature of the individual sample and thesample medium.

Therefore, in one aspect of the invention, for a particular heavymetal-binding compound of the present invention to be useful fordetection purposes, it must exhibit a detectable change in spectralproperties upon complexation of the desired metal ion (target ion) inthe chelating moiety. This is necessary when heavy metal-bindingcompounds complexed with heavy metals cannot be separated from heavymetal-binding compounds that are not bound to heavy metal ions.Preferably the change in spectral properties is a change in fluorescenceproperties. More preferably, the instant compounds display an intensityincrease or decrease in emission energy upon the complexation of thedesired target ion. Heavy metal-binding compounds that comprise areporter molecule that is colorimetric or fluorometric are hereinreferred to as “indicators”.

Alternatively, a change in spectral properties upon binding of a targetion is not necessary wherein a stable ternary complex is formed.Typically this ternary complex is immobilized allowing for the removalof unbound heavy metal-binding compounds.

In another embodiment, is provided a complex comprising a present metalion reporter molecule and a metal ion. To facilitate the detection ofthe metal ion-reporter molecule complex, the excitation or emissionproperties of the fluorescent complex are utilized.

For example, the sample is excited by a light source capable ofproducing light at or near the wavelength of maximum absorption of thefluorescent complex, such as an ultraviolet or visible lamp, an arclamp, a laser, or even sunlight. Preferably the fluorescent complex isexcited at a wavelength equal to or greater than about 300 nm, morepreferably equal to or greater than about 340 nm. The fluorescence ofthe complex is detected qualitatively or quantitatively by detection ofthe resultant light emission at a wavelength of greater than about 400nm, more preferably greater than about 450 nm, most preferred greaterthan 480 nm. The emission is detected by means that include visibleinspection, photographic film, or the use of current instrumentationsuch as fluorometers, quantum counters, plate readers, epifluorescencemicroscopes and flow cytometers or by means for amplifying the signalsuch as a photomultiplier.

In another embodiment is provided a method for detecting the presence orabsence of target ions in a live cell comprises the following steps:

-   -   a) combining a present metal ion reporter molecule with the        sample to prepare a labeling mixture wherein the metal ion        reporter molecule comprises at least one lipohilic group;    -   b) incubating the labeling mixture for a sufficient amount of        time for the metal ion reporter molecule to associate with the        metal ion in the live cell to form an incubated mixture;    -   c) illuminating the incubated sample with an appropriate        wavelength to form an illuminated mixture; and,    -   d) observing the illuminated mixture whereby the presence or        absence of the metal ion in a live cell is detected

Therefore, in an exemplary embodiment, compounds useful for detectingheavy metal ions in a live cell contain at least one—(CH₂OC(O)(CH₂)_(n)CH₃) group or lipophilic group. Typically, R_(X),R_(Y), and R_(X)′ are —CH₂CO₂R, wherein R is CH₂OC(O)(CH₂)_(n)CH₃.further aspect, the compound typically comprises a reporter moleculethat is a fluorophore wherein at least one of R¹-R⁸ is a fluorophore.Preferably R³ or R⁶ is a xanthene fluorophore. Alternatively, thefluorophore may be substituted by a lipophilic group including, but notlimited to, an AM ester.

In another embodiment is provided present heavy metal-binding compoundsthat are chemically reactive wherein the compound is covalently attachedto a reactive group. In this way the chemically reactive heavymetal-binding compounds can be conjugated to a desired reportermolecule, carrier molecule or solid support, which may be selected fromany of the above disclosed molecules and groups. The specific heavymetal-binding compound used in an assay or experiment is selected basedon the desired affinity for the target heavy metal ion as determined bythe expected concentration range in the sample, the desired end result,(e.g., binding, isolating or detecting), the desired live cellproperties and the desired selectivity. These chemically reactive heavymetal-binding compounds allow for the end user to tailor the compound totheir desired experiment.

In an exemplary embodiment, the present compounds comprise a carriermolecule or a solid support. In one aspect the carrier molecule or solidsupport facilitates the binding and sequestration of heavy metal ionsfrom a solution. This has utility wherein it is desirable to deplete asolution of heavy metal ions, such as the commercially available CalciumSponge (Molecular Probes, Inc.) for the removal of calcium ions fromsolution. The present compounds may be conjugated to any solid supportsuch that when a solution containing heavy metal ions comes into contactwith the present heavy metal-binding compounds the heavy metal ions arebound while the sample solution is allowed to pass freely by theimmobilized heavy metal-binding compounds. Once the heavy metal-bindingcompounds have been saturated, the heavy metal ions can be released fromthe heavy metal-binding compounds by combining with an appropriatebuffer to regenerate the heavy metal-binding conjugates. Appropriatebuffers that are useful for releasing heavy metal ions from the presentcompounds include solutions of tetrakis-(2-pyridylmethyl)ethylenediamine(TPEN). After the removal of the heavy metal ions, additional samplesolution may be passed over the immobilized heavy metal-bindingcompounds to further remove heavy metal ions.

In one aspect, the heavy metal-binding compounds are conjugated to apolymer such as a microparticle, dextran, agarose, acrylamide,polystyrene, see Example 6 Compound 25. These conjugates are useful topack into a column wherein a sample solution may be run through thecolumn to remove undesirable heavy metal-ions. These conjugates are alsouseful for isolating and concentrating heavy metal ions. In this way thecolumn may become saturated a number of times and heavy metal-ionsrepeatedly released to form a concentrated pool of heavy metal ions.

In another aspect, the present compounds are conjugated to a proteinsuch as an antibody. In this way the heavy metal-binding compounds areselectively localized to a target wherein heavy metal ions are bound andoptionally detected when the compound also comprises a reportermolecule.

In one embodiment, the compounds of the invention, in any of theembodiments described above, are associated, either covalently ornoncovalently, with a solid support and a reporter molecule. The solidsupport includes, without limitation, a microfluidic chip, a siliconchip, a microscope slide, a microplate well, or another solid matrix,wherein the metal ion binding/solid support/reporter molecule compoundis combined with the sample of interest as it flows over the surface.The detectable optical response is therefore detected on the matrixsurface itself, typically by use of an instrument. This embodiment ofthe invention is particularly suited to high-throughput screening and/orhigh content screening using automated methods, as disclosed in U.S.Pat. No. 6,127,133.

Sample Preparation

The sample is generally a representative cell population, fluid orliquid suspension that is known or suspected to contain the target ion.Representative samples include intracellular fluids such as in bloodcells, cultured cells, muscle tissue, neurons and the like;extracellular fluids in areas immediately outside of cells; in vesicles;in vascular tissue of plants and animals; in biological fluids such asblood, saliva, and urine; in biological fermentation media; inenvironmental samples such as water, soil, waste water and sea water; inindustrial samples such as pharmaceuticals, foodstuffs and beverages;and in chemical reactors. Detection and quantitation of the target ionin a sample can help characterize the identity of an unknown sample, orfacilitate quality control of a sample of known origin.

Thus, the sample can be a biological fluid such as whole blood, plasma,serum, nasal secretions, sputum, saliva, urine, sweat, transdermalexudates, cerebrospinal fluid, or the like. Biological fluids alsoinclude tissue and cell culture medium wherein an analyte of interesthas been secreted into the medium. Alternatively, the sample may bewhole organs, tissue or cells from the animal. Examples of sources ofsuch samples include muscle, eye, skin, gonads, lymph nodes, heart,brain, lung, liver, kidney, spleen, thymus, pancreas, solid tumors,macrophages, mammary glands, mesothelium, and the like. Cells includewithout limitation prokaryotic cells such as bacteria, yeast, fungi,mycobacteria and mycoplasma , and eukaryotic cells such as nucleatedplant and animal cells that include primary cultures and immortalizedcell lines. Typically prokaryotic cells include E. coli and S. aureus.Eukaryotic cells include without limitation ovary cells, epithelialcells, circulating immune cells, β cells, hepatocytes, and neurons.

In an exemplary embodiment, the sample comprises biological fluids,buffer solutions, live cells, fixed cells, eukaryotic cells, prokaryoticcells, nucleic acid polymers, nucleosides, nucleotides, a polymeric gelor tissue sections. In a further aspect, the sample comprises live cellsin an aqueous buffer.

The sample may be incubated in the presence of the nucleic acid reportermolecules for a time sufficient to form a nucleic acid-reporter moleculecomplex. While permeation and complexation may be more or less rapid forthe compounds disclosed herein, largely depending on the nature of thesubstituents present on the compound. It should be apparent to oneskilled in the art that the time necessary for sufficient permeation ofthe dye, or sufficient formation of the resulting nucleic acid complex,is dependent upon the physical and chemical nature of the individualsample and the sample medium (see for example U.S. Pat. No. 5,658,751).

Quantification of target ion levels in samples is typically accomplishedusing the indicators of the present invention by methods known in theart. For example, the ratiometric measurement of ion concentrationprovides accurate measurement of ion concentrations by the treatment ofthe fluorescence data as the ratio of excitation or fluorescenceintensities at two wavelengths, rather than the absolute intensity at asingle wavelength. Using the ratio method, a number of variables thatmay perturb the ion concentration measurements are eliminated. Inparticular, ion-dependent factors that affect the signal intensity, suchas nonuniform intracellular dye concentrations, probe leakage, dyebleaching and cell thickness, are canceled in the ratio measurements,since these parameters have a similar effect on intensities at bothwavelengths. While the ratio method can be used to determineconcentrations using observation of either the excitation spectra of theindicator, the emission spectra of the indicator, or both, in the caseof the indicators of the present invention, the shift in excitationenergy upon binding metal ions makes observation of the excitationspectrum a more useful technique. In either case, to achieve maximalutility, the indicator must be calibrated (to compensate for variance inthe dissociation constant of the indicator due to ionic strength,viscosity, or other conditions within the sample). To calibrate theindicator, ionophores such as A-23187, gramicidin, valinomycin, orionomycin are used. Non-ratiometric analysis can also be accomplished bycalibration with a second fluorescent dye present in the sample.

Illumination

The sample containing a metal ion-reporter molecule complex may beilluminated with a wavelength of light selected to give a detectableoptical response, and observed with a means for detecting the opticalresponse. By optical response is meant any detectable colorimetric orluminescent property of the complex. Typically, the optical response isrelated to the excitation or emission properties of the complex.

For example, the sample may be excited by a light source capable ofproducing light at or near the wavelength of maximum absorption of thefluorescent complex, such as an ultraviolet or visible lamp, an arclamp, a laser, or even sunlight. The optical response is optionallydetected by visual inspection, or by use of any of the followingdevices: CCD camera, video camera, photographic film, laser-scanningdevices, fluorometers, photodiodes, quantum counters, epifluorescencemicroscopes, scanning microscopes, flow cytometers, fluorescencemicroplate readers, or by means for amplifying the signal such asphotomultiplier tubes. Where the sample is examined using a flowcytometer, examination of the sample optionally includes sortingportions of the sample according to their fluorescence response.

The wavelengths of the excitation and emission bands of the metal ionreporter molecules vary with reporter molecule composition to encompassa wide range of illumination and detection bands. This allows theselection of individual reporter molecules for use with a specificexcitation source or detection filter. In particular, present reportermolecules can be selected that possess excellent correspondence of theirexcitation band with the 488 nm band of the commonly used argon laser oremission bands which are coincident with preexisting filters.

The presence, location, and distribution of metal ions, may be detectedusing the spectral properties of the compound-metal ion complex. Oncethe dye-metal ion complex is formed, its presence may be detected andused as an indicator of the presence, location, or type of metal ions inthe sample, or as a basis for sorting cells, or as a key tocharacterizing the sample or cells in the sample. Such characterizationmay be enhanced by the use of additional reagents, including fluorescentreagents.

The foregoing methods having been described it is understood that themany and varied compounds of the present invention can be utilized withthe many methods. The compounds not being limited to just those that arespecifically disclosed.

Therefore, in an exemplary embodiment, the present methods utilizecompounds having the formula:

Wherein R_(X), R_(Y) and R_(X)′ are each independently hydrogen, alkyl,substituted alkyl or —CR¹³R¹⁴CO₂R, wherein R is H, a salt ion or—CH₂OC(O)(CH₂)_(n)CH₃ and n is 0 to 6. Each R¹³ and R¹⁴ is independentlyhydrogen, alkyl or substituted alkyl. In one aspect, R_(X), R_(Y), orR_(X)′ are each hydrogen. In another aspect, are each R_(X), R_(Y), andR_(X)′ are —CH₂CO₂R wherein R is H, a salt ion or CH₂OC(O)CH₃. In afurther aspect, R is hydrogen or a salt ion. In yet another aspect, R isCH₂OC(O)CH₃.

R¹-R⁸ are independently hydrogen, halogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, hydroxyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, amino, substituted amino,alkylamino, substituted alkylamino, aldehyde, carboxyl, azido, nitro,nitroso, cyano, thioether, sulfoalkyl, carboxyalkyl, aminoalkyl,reporter molecule, reactive group, carrier molecule or solid support.Alternatively, any adjacent R¹-R⁸ together with the atoms to which theyare joined, form a ring which is a 5-, 6- or 7-memberedheterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-memberedcycloalkyl, a 5-, 6- or 7-membered heteroaryl, a substituted 5-, 6- or7-membered heteroaryl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6-or 7-membered aryl or reporter molecule.

The bridge substituents R⁹-R¹² are independently hydrogen, alkyl,substituted alkyl, reactive group, carrier molecule, or solid support.Alternatively, R⁹ in combination with R¹⁰; or R¹¹ in combination withR¹² together with the atoms to which they are joined, form a ring whichis a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or7-membered heterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, asubstituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheteroaryl, a substituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or7-membered aryl, or a substituted 5-, 6- or 7-membered aryl.

In one embodiment, the present compound comprises a reporter moleculethat is a chromophore, fluorophore, fluorescent protein, phosphorescentdye or a tandem dye. In one aspect, the reporter molecule is a xanthene,indole, cyanine, oxazole, dansyl, borapolyazaindacene, benzofuran,quinazolinone, benzazole, oxazine, pyrene, naphthalene, coumarin,biotin, enzyme substrate or fluorescent protein. In a further aspect,the xanthene is a fluorescein or derivative thereof, rhodamine orderivative thereof, rhodol or derivative thereof or rosamine or aderivative thereof. In another further aspect, the reporter molecule isoptionally and independently substituted by hydrogen, halogen, amino,substituted amino, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, sulfo, lipophilic group (AMester or acetate ester) solid support, reactive group or carriermolecule. In one embodiment, at R⁸ is a reporter molecule. In oneaspect, at least one of R³ or R⁶ is a reporter molecule.

In another exemplary embodiment, the compounds of the methods comprise areactive group, solid support or carrier molecule either with or withoutthe compound further comprising a reporter molecule. The reactive group,solid support, carrier molecule and optionally the reporter moleculecomprise a linker that is a single covalent bond, or a covalent linkagethat is linear or branched, cyclic or heterocyclic, saturated orunsaturated, having 1-20 nonhydrogen atoms selected from the groupconsisting of C, N, P, O and S; and are composed of any combination ofether, thioether, amine, ester, carboxamide, sulfonamide, hydrazidebonds and aromatic or heteroaromatic bonds. Alternatively, the reportergroup, such as benzofuran, does not comprise a linker, but insteadshares atoms with the ring A or ring B of the acetic acid analog ofBAPTA.

Thus, in one embodiment of the methods the compounds comprise a reactivegroup that is useful for forming conjugates to reporter molecules, solidsupports and/or carrier molecules. The reactive groups are selected fromthe group consisting of an acrylamide, an activated ester of acarboxylic acid, a carboxylic ester, an acyl azide, an acyl nitrile, analdehyde, an alkyl halide, an anhydride, an aniline, an amine, an arylhalide, an azide, an aziridine, a boronate, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazine, an imido ester, anisocyanate, an isothiocyanate, a maleimide, a phosphoramidite, areactive platinum complex, a silyl halide, a sulfonyl halide, a thioland a photoactivatable group. In one aspect, the reactive group isselected from the group consisting of carboxylic acid, succinimidylester of a carboxylic acid, hydrazide, amine and a maleimide.

In another embodiment of the methods the compounds comprise a carriermolecule selected from the group consisting of an amino acid, a peptide,a protein, a polysaccharide, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, ahormone, a lipid, a lipid assembly, a synthetic polymer, a polymericmicroparticle, a biological cell or a virus. In one aspect, the carriermolecule is selected from the group consisting of an antibody orfragment thereof, an avidin or streptavidin, a biotin, a dextran, an IgGbinding protein, a fluorescent protein, agarose, and a non-biologicalmicroparticle.

In yet another embodiment of the methods, the compound comprise a solidsupport selected from the group consisting of a microfluidic chip, asilicon chip, a microscope slide, a microplate well, silica gels,polymeric membranes, particles, derivatized plastic films, glass beads,cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride,polypropylene, polyethylene, nylon, latex bead, magnetic bead,paramagnetic bead, and superparamagnetic bead. In one aspect, the solidsupport is selected from the group consisting of Sepharose,poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar,cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin,mannan, inulin, nitrocellulose, diazocellulose and starch.

Kits

Suitable kits for forming a metal ion-reporter molecule complex anddetecting the metal ions also form part of the present disclosure. Suchkits can be prepared from readily available materials and reagents andcan come in a variety of embodiments. The contents of the kit willdepend on the design of the assay protocol or reagent for detection ormeasurement. All kits will contain instructions, appropriate reagents,and one or more of the presently disclosed nucleic acid reportermolecules. Typically, instructions include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter such as the relative amounts of reagent and sample to be addedtogether, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like to allow the user to carryout any one of the methods or preparations described above. In oneaspect, the kit is formulated to facilitate the high-throughputscreening of multiple samples, such as may be accomplished usingautomated methods.

Therefore, kits of the present invention comprise at least one heavymetal-binding compound of the present invention in an appropriatestorage form, e.g. lyophilized or dissolved in an organic solvent, andinstructions for preparing the heavy metal-binding compound to be usedby the end user.

In addition, the kits may contain appropriate controls (including apositive control), metal ion calibration standards, ample preparationreagents, an aqueous metal ion reporter molecule dilution buffer, anorganic solvent, and additional detection reagents such as calcium ionindicators, organelle stains, a metal ion indicator other than for heavymetal ions, an antibody or fragment thereof or a reference dye standard.

Therefore, in an exemplary embodiment, the kits contain compounds havingthe formula:

Wherein R_(X), R_(Y) and R_(X)′ are each independently hydrogen, alkyl,substituted alkyl or —CR¹³R¹⁴CO₂R, wherein R is H, a salt ion or—CH₂OC(O)(CH₂)_(n)CH₃ and n is 0 to 6. Each R¹³ and R¹⁴ is independentlyhydrogen, alkyl or substituted alkyl. In one aspect, R_(X), R_(Y), orR_(X)′ are each hydrogen. In another aspect, are each R_(X), R_(Y), andR_(X)′ are —CH₂CO₂R wherein R is H, a salt ion or CH₂OC(O)CH₃. In afurther aspect, R is hydrogen or a salt ion. In yet another aspect, R isCH₂OC(O)CH₃.

R¹-R⁸ are independently hydrogen, halogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, hydroxyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, amino, substituted amino,alkylamino, substituted alkylamino, aldehyde, carboxyl, azido, nitro,nitroso, cyano, thioether, sulfoalkyl, carboxyalkyl, aminoalkyl,reporter molecule, reactive group, carrier molecule or solid support.Alternatively, any adjacent R¹-R⁸ together with the atoms to which theyare joined, form a ring which is a 5-, 6- or 7-memberedheterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-memberedcycloalkyl, a 5-, 6- or 7-membered heteroaryl, a substituted 5-, 6- or7-membered heteroaryl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6-or 7-membered aryl or reporter molecule.

The bridge substituents R⁹-R¹² are independently hydrogen, alkyl,substituted alkyl, reactive group, carrier molecule, or solid support.Alternatively, R⁹ in combination with R¹⁰; or R¹¹ in combination withR¹² together with the atoms to which they are joined, form a ring whichis a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or7-membered heterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, asubstituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-memberedheteroaryl, a substituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or7-membered aryl, or a substituted 5-, 6- or 7-membered aryl.

In one embodiment, the present compound comprises a reporter moleculethat is a chromophore, fluorophore, fluorescent protein, phosphorescentdye or a tandem dye. In one aspect, the reporter molecule is a xanthene,indole, cyanine, oxazole, dansyl, borapolyazaindacene, benzofuran,quinazolinone, benzazole, oxazine, pyrene, naphthalene, coumarin,biotin, enzyme substrate or fluorescent protein. In a further aspect,the xanthene is a fluorescein or derivative thereof, rhodamine orderivative thereof, rhodol or derivative thereof or rosamine or aderivative thereof. In another further aspect, the reporter molecule isoptionally and independently substituted by hydrogen, halogen, amino,substituted amino, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, sulfo, lipophilic group (AMester or acetate ester) solid support, reactive group or carriermolecule. In one embodiment, at least one of R¹- R⁸ is a reportermolecule. In one aspect, at least one of R³ or R⁶ is a reportermolecule.

In another exemplary embodiment, the compounds of the kits comprise areactive group, solid support or carrier molecule either with or withoutthe compound further comprising a reporter molecule. The reactive group,solid support, carrier molecule and optionally the reporter moleculecomprise a linker that is a single covalent bond, or a covalent linkagethat is linear or branched, cyclic or heterocyclic, saturated orunsaturated, having 1-20 nonhydrogen atoms selected from the groupconsisting of C, N, P, O and S; and are composed of any combination ofether, thioether, amine, ester, carboxamide, sulfonamide, hydrazidebonds and aromatic or heteroaromatic bonds. Alternatively, the reportergroup, such as benzofuran, does not comprise a linker, but insteadshares atoms with the ring A or ring B of the acetic acid analog ofBAPTA.

Thus, in one embodiment of the kits the compounds comprise a reactivegroup that is useful for forming a conjugate with a reporter molecule,carrier molecule and/or solid support. The reactive group is selectedfrom the group consisting of an acrylamide, an activated ester of acarboxylic acid, a carboxylic ester, an acyl azide, an acyl nitrile, analdehyde, an alkyl halide, an anhydride, an aniline, an amine, an arylhalide, an azide, an aziridine, a boronate, a diazoalkane, ahaloacetamide, a halotriazine, a hydrazine, an imido ester, anisocyanate, an isothiocyanate, a maleimide, a phosphoramidite, areactive platinum complex, a silyl halide, a sulfonyl halide, a thioland a photoactivatable group. In one aspect, the reactive group isselected from the group consisting of carboxylic acid, succinimidylester of a carboxylic acid, hydrazide, amine and a maleimide.

In another embodiment of the kits the compounds comprise a carriermolecule selected from the group consisting of an amino acid, a peptide,a protein, a polysaccharide, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, ahormone, a lipid, a lipid assembly, a synthetic polymer, a polymericmicroparticle, a biological cell or a virus. In one aspect, the carriermolecule is selected from the group consisting of an antibody orfragment thereof, an avidin or streptavidin, a biotin, a dextran, an IgGbinding protein, a fluorescent protein, agarose, and a non-biologicalmicroparticle.

In yet another embodiment of the kits, the compound comprise a solidsupport selected from the group consisting of a microfluidic chip, asilicon chip, a microscope slide, a microplate well, silica gels,polymeric membranes, particles, derivatized plastic films, glass beads,cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride,polypropylene, polyethylene, nylon, latex bead, magnetic bead,paramagnetic bead, and superparamagnetic bead. In one aspect, the solidsupport is selected from the group consisting of Sepharose,poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar,cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin,mannan, inulin, nitrocellulose, diazocellulose and starch.

A detailed description of the invention having been provided above, thefollowing examples are given for the purpose of illustrating theinvention and shall not be construed as being a limitation on the scopeof the invention or claims.

Examples Example 1 Preparation of Compound 12

Compounds 1 (11.3 g, 36.8 mmol) and 2 (5.35 g, 38.5 mmol) were dissolvedin DMF (˜200 mL), then potassium carbonate (13.8 g, 100 mmol) was added.The reaction mixture was heated at 110-130° C. with stirring for 12-18hours, until TLC (R_(f)=0.6, EtOAc/hexanes (30:70)) of a water-EtOAcaliquot shows that the starting material had been consumed. The reactionmixture was cooled to ambient temperature, then poured into ice water(˜1.2 L) and stirred at ambient temperature. After stirring for ˜15minutes the oil coagulated into a crystalline solid. Stirring wascontinued until a filterable solid was obtained. The solids werecollected on a glass frit funnel by suction-filtration, rinsed withwater, and dried to yield 12.7 g of 3 as pale yellow crystalline solid.

A solution of 3 (12.7 g, 34.76 mmol) in 1:1 chloroform/acetic acid (˜100mL) was treated with 10% palladium on carbon (1.0 g) and shaken with aParr shaker under hydrogen at ˜40 psi for 12-18 hours, until TLC(R_(f)=0.6, EtOAc/hexanes (30:70)) showed consumption of 3. The reactionmixture was suction filtered through Celite in a Buchner funnel, rinsingthe solid with chloroform. The filtrate was concentrated with rotaryevaporation to give an off-white solid. The solid was washed withhexanes and dried to yield 8.35 g of 4 as colorless solid.

A solution of 4 (0.51 g, 2.1 mmol), methyl bromoacetate (3.0 mL, d1.616, 32 mmol) and diisopropylethylamine (1.74 mL, d 0.742, 10 mmol) inacetonitrile (˜25 mL) was heated at reflux under argon with a condenserand magnetic stirring for ˜48 hours, until TLC (EtOAc/hexanes (3:2))showed conversion of 4 (R_(f)=0.7) into 5 (R_(f)=0.6). The volatileswere removed with rotary evaporation. The residue was dissolved in EtOAc(˜75 mL) then washed with 10% citric acid (2×25 mL) and brine (1×25 mL).The organic layer was dried over sodium sulfate, filtered andconcentrated to a brown residue. This residue was purified by flashchromatography on silica gel (37×5 cm column) using increasing amountsof ethyl acetate in hexanes. Pure product fractions were pooled anddried by rotary evaporaton to give 5 as 485 mg as pale brown foam.

A solution of 5 (0.60 g, 1.3 mmol) in dry DMF (5 mL) under argon wastreated with phosphorous oxychloride (0.49 mL, d 1.645, 5.2 mmol) atambient temperature. The resulting clear solution was stirred for 12-18hours in a 40° C. bath, until TLC (EtOAc/hexanes (3:2)) of a water-EtOAcaliquot showed consumption of 5 (R_(f)=0.6) and formation of 6(R_(f)=0.45). The reaction solution was cooled to ambient temperaturethen poured into water (˜150 mL). The pH was adjusted to 7 with aqueoussodium carbonate. The resulting precipitate was collected on a Buchnerfunnel, rinsed with water and dried under house vacuum to yield 0.61 gof 6 as light yellow powder.

A solution of 6 (200 mg, 0.41 mmol) and 7 (105 mg, 0.82 mmol) inmethanesulfonic acid (˜4 mL) was stirred at ambient temperature with amagnetic stir bar for ˜30 min., until TLC (CHCl₃/MeOH/AcOH (20:4:1)) ofa water-EtOAc aliquot showed consumption of 6 (R_(f)=0.9) and 7(R_(f)=0.45), and formation of 8 (R_(f)=0.2). The reaction solution wasadded dropwise into a stirring solution of 3M sodium acetate (˜40 mL).The resulting precipitate was collected on a Buchner funnel, rinsed withwater, and dried vacuum to yield 0.28 g of 8 as colorless powder.

p-Chloranil (0.37 gm 1.52 mmol) was added to a solution of 8 (0.54 g,0.76 mmol) in CHCl₃/MeOH (1:1, ˜30 mL). The orange reaction mixture wasstirred at reflux 12-18 hours, until TLC (CHCl₃/MeOH/AcOH (50:5:1))showed formation of 9 (R_(f)=0.75, orange spot). The reaction mixturewas cooled to ambient temperature, and the solvents removed with rotaryevaporation. The residue was purified by flash chromatography on silicagel (4×28 cm column) using CHCl₃/MeOH/AcOH (50:5:1) as the elutionsolvent. Desired fractions were combined and concentrated in vacuo togive 9 as 0.30 g of a dark orange solid.

A solution of 9 (0.30 g, 0.42 mmol) in methanol/dioxane (1:1, 20 mL) wastreated with 1M KOH (4.5 mL, 4.5 mmol). The reaction mixture was stirredfor 12-18 hours, until TLC (dioxane/IPA/H₂O/NH₄OH (15:58:13:14)) showedconsumption of 9 (R_(f)=0.95) and formation of 10 (R_(f)=0.4, orangespot). The pH was adjusted from 12.0, as measured by a pH meter, to 9.0by dropwise addition of 1M HCl. The solvents were removed with a rotaryevaporation to a dry residue. This residue was purified bychromatography on Sephadex LH-20 (4×37 cm column) using water as eluant.Desired product fractions were pooled and lyophilized to give 10 as (245mg of an orange powder.

The pH of a solution of 10 (25 mg, 0.031 mmol) in H₂O (˜3 mL) wasadjusted from 7.5 to 2.0 by dropwise addition of 5% aqueous HCl withstirring. The resulting precipitate was collected on a Hirsch funnel,rinsed with water, and dried under vacuum to yield 21 mg of 11 as anamber powder. A solution of 11 (35 mg, 0.053 mmol) in dry DMF (˜2 mL)was prepared under argon, then bromomethyl acetate (50 μL, d 1.560, 0.53mmol) and diisopropylethylamine (92 ∞L, d 0.742, 0.53 mmol) were added.The orange reaction solution was stirred at ambient temperature for ˜3hours, until TLC (MeOH/CHCl₃ (10:90)) of a water-EtOAc aliquot showedthe reaction to be complete (Rf=0.7, orange dim fluorescent spot). Thereaction mixture was poured into 10% aqueous citric acid (˜40 mL). Theresulting mixture was extracted with 20% toluene/EtOAc (2×25 mL). Theorganic layer was washed with water (1×20 mL) and brine (1×20 mL), driedover sodium sulfate, filtered and concentrated with rotary evaporationto yield 52 mg of crude 12 as an orange residue. This residue waspurified by flash chromatography on a silica gel column (2×26 cm) using10% methanol/chloroform as eluant. Pure product fractions were pooledand concentrated to give 12 as 25 mg of an orange solid.

Example 2 Preparation of Compound 15

A pale brown mixture of compound 6 (0.20 g, 0.41 mmol) and crystallized3-dimethylaminophenol (0.11 g, 0.82 mmol) in 6 mL propionic acid washeated overnight at 60° C. under argon. TLC (EtOAc/hexanes 3:2) of awater/EtOAc aliqhot showed consumption of 6 (Rf 0.55) and formation ofcompound 13 (Rf 0.20). After cooling, the reaction solution was addedslowly to 50 mL 3M aqueous sodium acetate with stirring. The resultingprecipitate was collected on a Buchner funnel, rinsed with water (3×10mL), and dried in vacuo to give 13 as 0.26 g of a lavender powder.

To a solution of compound 13 (0.26 g, 0.36 mmol) in 1:1methanol/chloroform (15 mL) was added p-chloranil (0.13 g, 0.54 mmol) atrt. The resulting mixture was stirred for 4 h, whereupon TLC(chloroform/methanol/acetic acid, 50:5:1) showed consumption of 13 (Rf0.25), and formation of compound 14 (Rf 0.05, red fluorescent spot). Thevolatiles were removed in vacuo, and the residue purified by flashchromatography on a silica gel column using chloroform/methanol/aceticacid (50:5:1) as eluant. Fractions containing pure 14 were pooled andconcentrated in vacuo to give 0.13 g of a purple solid.

To a red solution of compound 14 (0.13 g, 0.18 mmol) in 1:1methanol/dioxane (11 mL) was added a 1M solution of KOH (1.5 mL, 1.5mmol). The resulting pale red-brown mixture was stirred at rt, whereuponafter 3h TLC (dioxane-isopropanol-water-ammonium hydroxide(15:58:13:14)) showed consumption of 14 (Rf 0.75) and formation of 15(Rf 0.25). The reaction pH was lowered from 13 to 8 by dropwise additionof 10% aqueous citric acid, followed by concentration in vacuo. Theresidue was purified by gravity chromatography on Sephadex LH-20 usingwater as eluant. Pure product fractions were combined and lyophilized togive compound 15 as 79 mg of red powder.

Example 3 Preparation of Compound 18

To a light yellow solution of compound 6 (0.18 g, 0.37 mmol) andphosphonium salt 7 (0.20 g, 0.37 mmol) in dry DMF (8 mL) under argon wasadded potassium carbonate (72 mg, 0.52 mmol). The resulting mixture washeated at 90° C. for 48 h, whereupon TLC (EtOAc/hexanes 3:2) showedconsumption of 6 (Rf 0.50)and formation of stilbene 16 (Rf 0.60). Aftercooling, the reaction mixture was poured into 80 mL 10% citric acid,then extracted with 4:1 EtOAc/toluene (4×25 mL). The extract was washedwith water (1×) and brine (1×), dried over sodium sulfate, andconcentrated in vacuo to give crude compound 16 as 0.48 g of ared-yellow oil. Further purification was effected by flashchromatography on silica gel using 1:1 EtOAc/hexanes. Pure productfractions were combined and evaporated to give 16 as 0.19 g of ared-yellow oil.

A solution of compound 16 (0.19 g, 0.29 mmol) in triethylphosphite (3mL) was heated to 120° C. under argon for 16 h, whereupon TLC(EtOAc/hexanes 3:2) showed conversion of compound 16 (Rf 0.55) intocompound 17 (Rf 0.45, blue fluorescent). The volatiles were removed invacuo, and the residue purified by flash chromatography on silica gelusing 1:1 EtOAc/hexanes as eluant. Pure product fractions were combinedand evaporated to give compound 17 as 0.13 g of a pale yellow immobileglass.

To a solution of compound 17 (0.13 g, 0.20 mmol) in 5 mLdioxane/methanol (1:1) was added a 1M KOH solution (1.4 mL, 1.4 mmol).The resulting solution was kept at rt for 2 h, whereupon TLC(dioxane-isopropanol-water-ammonium hydroxide (15:58:13:14)) showedconversion of 17 (Rf 0.9) to compound 18 (Rf 0.4). The volatiles wereremoved in vacuo, and the residue purified by chromatography on aSephadex LH-20 column using water as eluant. Pure product fractions werecombined and lyophilized to give compound 18 as 49 mg of a pale yellowpowder.

Example 4 Preparation of Compound 25

A 0.1 M solution of aldehyde 6 in acetic anhydride is cooled on ice, andtreated dropwise with 1.05 eq 70% nitric acid with stirring. The icebath is removed and the resulting dark yellow solution is stirred untilTLC shows complete nitration (˜30 minutes). The reaction solution ispoured into excess water, followed by extraction with ethyl acetate. Theextract is washed with aqueous sodium bicarbonate, water, and brine,dried over sodium sulfate, and concentrated in vacuo to a red-yellow oilwhich is purified by flash chromatography on silica gel using increasingethyl acetate in hexanes to afford pure compound 19 as a yellow solid.

A 0.3 M solution of aldehyde 19 in methanesulfonic acid at rt is treatedwith 2 eq of 4-fluororesorcinol. The resulting mixture is stirred untilhomogeneity is achieved (˜30 minutes), then the reaction solution ispoured into excess water. The resulting precipitate is collected on aBuchner funnel, washed with copious amounts of water, and dried in vacuoto give compound 20 as an off-white powder.

A 0.2 M solution of nitro compound 20 in methanol is treated with 10wt %of 10% palladium/carbon. The resulting mixture is shaken overnight in aParr apparatus at 40 psi hydrogen gas. The resulting mixture is suctionfiltered through Celite, and concentrated in vacuo to give aniline 21 asa pale brown glass.

A 0.5 M solution of aniline 21 in dichloromethane is treated with 20 eqof trifluoroacetic anhydride. The resulting solution is stirred for 3hours at rt, then poured into excess water. The organic phase is washedwith aqueous sodium bicarbonate (3×), water, and brine, dried oversodium sulfate, and concentrated in vacuo to give trifluoroacetamide 22as a pale brown glass.

A 0.2 M solution of trifluoroacetamide 22 in 1:1 chloroform/methanol istreated with 2 eq of p-chloranil. The resulting mixture is heated atreflux for 48 hours. After cooling, the reaction mixture is filtered andthe trifluoroacetamide 23 is isolated by flash chromatography on silicagel using increasing amounts of methanol in chloroform. Compound 23 isobtained as an amber powder.

A 0.5 M solution of 23 in methanol is treated with aqueous potassiumcarbonate (5 eq) at rt. The resulting solution is stirred overnight,then concentrated in vacuo. The residue is taken up in 10% aqueouscitric acid, then extracted 3× with chloroform. The extract is driedover sodium sulfate and concentrated in vacuo. The resulting aniline isimmediately dissolved in chloroform at 0.5 M, then treated with 10 eqthiophosgene. The resulting solution is stirred for 3 hours, thenconcentrated in vacuo. Toluene (2×) is stripped from the residue, whichis dried under high vacuum overnight to give compound 24.

A 0.5M solution of compound 24 in DMSO is added to a 0.2 M solution of10000 MW aminodextran in aqueous sodium bicarbonate, in a ratio of 5 eqof Compound 24 per amino group per dextran. The reaction pH is 8-9. Theresulting mixture is stirred for 48 hr at rt, then added dropwise to a50× volume excess of methanol. The resulting precipitate is collected bycentrifugation and washed with fresh methanol, then dried in vacuo togive an orange powder. This powder (the tetramethyl ester of compound25) is dissolved in water to 0.5 M, then treated with aq KOH so that thereaction pH is 13. The resulting solution is kept overnight, then the pHis lowered to 9 and the volatiles removed in vacuo. The residue isapplied to a Sephadex LH-20 column as a solution in minimal water, theneluted with water. Pure product fractions are combined and lyophilizedto give compound 25 as a fluffy orange powder.

Example 5 Detection of Lead, Mercury and Cadmium Ions in Cuvettes UsingCompound 10

A dye stock (709 μM) solution of Compound 10 was prepared in DMSO. Lead,cadmium and mercury solutions from 0-100 μM were prepared in disposablecuvettes by diluting a 100 μM metal stock solutions to a final volume of2 mL with 50 mM MOPS (pH7). The 100 μM metal stocks were also in 50 mMMOPS (pH7). For the cadmium titration, dye stock was added to give afinal concentration of 0.35 μM. For the lead and mercury titrations, dyestock was added to give a final concentration of 1.42 μM. Samples wereexcited at 492 nm and fluorescence was recorded at 517 nm. The followingKd values were calculated: cadmium 1.3 μM, lead 4.9 μM, mercury 75 μM.See, FIG. 2.

Example 6 Detection of Lead, Cadmium and Mercury Ions in Cuvettes UsingCompound 15

The experiment was performed as described above except that the exactdye concentrations were unknown. Sample was dissolved in DMSO anddiluted 2× in nano-pure H₂O before running. Ex./Em. 549/577nm. Kdvalues: cadmium 4.6 uM, lead 2.1 μM, mercury 78 μM. See, FIG. 1.

Example 7 Detection of Cadmium Ions in Cuvettes Using Compound 18

The experiment was performed as described above for cdmium ion detectionexcept that the exact dye concentrations were unknown. Ex./Em.348/397/481 nm (ratiometric). Kd value: 1.2 μM.

Example 8 Detection of Intracellular Lead Ions

Jurkat cells were suspended at 1×10⁶/ml in saline (0.85% sodiumchloride), and 1 μM of Compound 12 was added to 1 ml of cells, incubatedat 37° C. for 1 hr, washed once with saline, and resuspended in eithersaline or 1 μM PbCl₂. 1 μM lonomycin was added to designated tubes,tubes were incubated for 30 minutes at 37° C., washed ×2 in saline.

The cell pellet was resuspended in saline, and tubes were run on a flowcytometer (Beckman Coulter Epics Elite ESP, Miami, Fla., USA). The 488nm laser was used for excitation, and single color fluorescence wascollected with 525/20BP emission filter. Tubes 1 thru 7 are all negativecontrols, with tube 8 the only one expected to have fluorescence. Tube1: Cells in saline only; Tube 2: cells in saline+1 μM ionomycin; Tube 3:cells in 1 μM PbCl₂ in saline; Tube 5: cells in 1 μM PbCl₂ in saline+1μM ionomycin; Tube 6: cells in saline+1 μM Compound 12+1 μM ionomycin;Tube 7: cells in 1 μM PbCl₂ in saline+1 μM Compound 12; Tube 8: cells in1 μM PbCl₂ in saline+1 μM Compound 12+1 μM ionomycin.

The first seven tubes were negative controls and the resulting MeanFluorescence Intensity (MFI) for all tubes was the same as unstainedcells (MFI range 2.7-3.7), See FIG. 4. The last sample, tube 8,containing Compound 12+PbCl₂+ionomycin, produced a positive response(MFI=77) that was easily distinguished from negative peaks. Theseresults demonstrate that the present Compound 12 was selectively bindingintracellular lead ions.

Example 9 Compound 12 Tested with PbCl₂ (1 nM-1 μM)

Jurkat cells were suspended at 1×10⁶/ml in saline (0.85% sodiumchloride), and 400 nM Compound 12 was added to 1 ml cells, incubated at37° C. for 1 hr, washed once with saline, and resuspended in eithersaline or a range of PbCl₂ solutions of 1 nM-1 μM 1 μM lonomycin wasadded to tubes, incubated 30 minutes at 37° C., and washed ×2 in saline.The cell pellet was resuspended in saline, and analyzed on a flowcytometer. The 488 nm laser was used for excitation, a gate was made onForward Scatter vs Side Scatter, and single color fluorescence wascollected with 525/20BP emission filter. The results demonstrate thatfluorescence intensity can be correlated to concentration of lead ionsand that 1 nm of PbCl₂ is distinguished from background fluorescence,See FIG. 5.

Example 10 Dual Staining with Propidium Iodide (to Distinguish DeadCells) and Compound 12 (to Detect Lead Ions)

Jurkat cells were suspended at 1×10⁶/ml in saline (0.85% sodiumchloride), and 1 μM Compound 12 was added to 1 ml cells, incubated at37° C. for 1 hr, washed once with saline, and resuspended in eithersaline or 1 μM PbCl₂. 1 μM ionomycin was added to tubes, incubated 30minutes at 37° C., and washed ×2 in saline. The cell pellet wasresuspended in saline. Propidium Iodide was added at 150 μM, incubatedfor 5 min at room temperature, and samples were run on a flow cytometer.The 488 nm laser was used for excitation, and dual color fluorescencewas collected with 525/20BP emission filter for Compound 12 and 610/20BPemission filter for PI. The results demonstrate that the population ofcells stained with propidium iodide (dead cells) are mutually exclusivefrom the population of cells stained with Compound 12 (live cells)demonstrating that Compound 12 is staining viable cells only, and doesnot cause cell death, See FIG. 6.

Example 11 Compound 12 Does Not Produce a Detectable Response in thePresence of Intracellular Calcium Ions

Jurkat cells were suspended at 1×10⁶/ml in saline (0.85% sodiumchloride). 1 μM Compound 12 was added to 1 ml cells, incubated at 37° C.for 1 hr, washed once with saline, and resuspended in 10 different FreeCalcium ion solutions (range 0-100 μM Calcium). 1 μM lonomycin was addedto tubes, incubated for 30 minutes at 37° C., and washed ×2 in saline.The cell pellet was resuspended in saline and analyzed on a flowcytometer. The 488 nm laser was used for excitation, with a live cellgate on Forward Scatter vs Side Scatter, and single color fluorescencewas collected with 525/20BP emission filter. No fluorescence responsewas seen with cells incubated with any concentration ofCalcium+ionomycin, See FIG. 7.

Example 12 Detection of Intracellular Cadmium Ions

Jurkat cells were suspended at 1×10⁶/ml in saline (0.85% sodiumchloride), and 400 nM Compound 12 was added to 1 ml cells, incubated at37° C. for 1 hr, washed once with saline, and resuspended in eithersaline or 1 μM, 500 nM, or 250 nM CdCl₂ 1 μM ionomycin was added to thesamples, incubated for 30 minutes at 37° C., and washed ×2 in saline.The cell pellet was resuspended in saline and analyzed on a flowcytometer. The 488 nm laser was used for excitation, a gate was made onForward Scatter vs Side Scatter, and single color fluorescence wascollected with 525/20BP emission filter. The results demonstrate thatthe present compound 12 binds and detects 1 μM, 500 nM, and 250 nMconcentrations of cadmium ions, See FIG. 8.

Example 13 Dual Staining with Propidium Iodide (to Distinguish DeadCells) and Compound 12 (to Detect Cadmium Ions)

Jurkat cells were suspended at 1×10⁶/ml in saline (0.85% sodiumchloride), and 1 μM Compound 12 was added to 1 ml cells, incubated at37° C. for 1 hr, washed once with saline, and resuspended in eithersaline or 100 μM CdCl₂ 1 μM lonomycin was added to the samples,incubated for 30 minutes at 37° C., and washed ×2 in saline. The cellpellet was resuspended in saline, Propidium Iodide was added at 150 μM,incubated for 5 min at room temperature, and tubes were analyzed on theflow cytometer. The 488 nm laser was used for excitation, and dual colorfluorescence was collected with 525/20BP emission filter for Compound 12and 610/20BP emission filter for propidium iodide. The resultsdemonstrate that Compound 12 is staining viable cells only and does notcause cell death, See FIG. 9.

Example 14 Compound 12 Tested with CdCl₂ (5-500 μM)

Jurkat cells were suspended at 1×10⁶/ml in saline (0.85% sodiumchloride), and 400 nM Compound 12 was added to 1 ml cells, incubated at37° C. for 1 hr, washed once with saline, and resuspended in eithersaline or a range of CdCl₂ solutions of 5-500 nM. 1 μM lonomycin wasadded to the samples, incubated 30 minutes at 37° C., and washed ×2 insaline. The cell pellet was resuspended in saline and analyzed on a flowcytometer. The 488 nm laser was used for excitation, a gate was made onForward Scatter vs Side Scatter, and single color fluorescence wascollected with 525/20BP emission filter. The results demonstrateincreasing fluorescence with increasing CdCl₂ concentration and that 5μM CdCl₂ is distinguished from the background fluorescence, See FIG. 10.

Example 15 Detection of Intracellular Lead Ions in Human Red Blood Cellswith Compound 12

Sodium Heparin anticoagulated whole blood was washed twice in warmedsaline, 1 ml RBCs in saline at 1×10⁶ cells/ml was added to tubes, 500 nMCompound 12 was added, cells were incubated at 37° C. for 60 minutes,protected from light, and washed with saline. The cell pellet wasresuspended in either saline or 1 μM PbCl₂+/−ionomycin. Cells incubatedwith Compound 12 in the presence of 1 μM PbCl₂ and 1 μM ionomycin gavefluorescence (MFI=57.3) distinguished from the negative controls (MFIrange 2.6-13.6), See FIG. 11.

Example 16 Testing of Compound 12 on 3T3 Cells with Both CadmiumChloride Solution and Lead Chloride Solution

Testing was performed using adherent cells 3T3 with Compound 12 atconcentration range 1 μM. 500 nM, 250 nM and 125 nM of PbCl₂ and atconcentration range 100uM, 50 μM and 25 μM of CdCl₂. The 3T3 cells weresuspended at 5×10⁶/ml in saline (0.85% sodium chloride), and 400 nMCompound 12 was added to 1 ml cells, incubated at 37° C. for 1 hr,washed once with saline, and resuspended in either saline or a range ofCdCl₂ and PbCl₂ solutions. 1 μM lonomycin was added to tubes, incubatedfor 30 minutes at 37° C., and washed ×2 in saline. The cell pellet wasresuspended in saline, Propidium Iodide was added at 150 μM, incubatedfor 5 min at room temperature, and the samples were analyzed on a flowcytometer. The 488 nm laser was used for excitation, and dual colorfluorescence was collected with 525/20BP emission filter for Compound 12and 610/20BP emission filter for Propidium Iodide.

After fluorescence acquisition, TPEN, a cell-permeant heavy metalchelator, was added to the samples. TPEN was used to chelate the heavymetal, demonstrated by drop of fluorescence after TPEN addition.

The preceding examples can be repeated with similar success bysubstituting the specifically described heavy metal-binding compoundsand heavy metal binding conditions of the preceding examples with thosegenerically and specifically described in the forgoing description. Oneskilled in the art can easily ascertain the essential characteristics ofthe present invention, and without departing from the spirit and scopethereof, can make various changes and modifications of the invention toadapt to various usages and conditions.

All patents and patent applications mentioned in this specification areherein incorporated by reference to the same extent as if eachindividual patent or patent application was specifically andindividually indicated to be incorporated by reference.

1.-66. (canceled)
 67. A kit for detecting metal ions in a sample,wherein the kit comprises a compound having the formula:

wherein R_(X) is —CH₂CO₂R; R_(Y) is —CH₂CO₂R; R_(X)′ is —CH₂CO₂R;wherein R is H, a salt ion or —CH₂OC(O)(CH₂)—CH₃ and n is 0 to 6; R¹ ishydrogen; R² is hydrogen; R³ is hydrogen, halogen, alkyl, substitutedalkyl, alkoxy, substituted alkoxy, hydroxyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, amino, substituted amino,alkylamino, substituted alkylamino, aldehyde, carboxyl, azido, nitro,nitroso, cyano, thioether, sulfoalkyl, carboxyalkyl, aminoalkyl,reporter molecule, reactive group, carrier molecule or solid support; R⁴is hydrogen; R⁵ is hydrogen; R⁶ is hydrogen, halogen, alkyl, substitutedalkyl, alkoxy, substituted alkoxy, hydroxyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, amino, substituted amino,alkylamino, substituted alkylamino, aldehyde, carboxyl, azido, nitro,nitroso, cyano, thioether, sulfoalkyl, carboxyalkyl, aminoalkyl,reporter molecule, reactive group, carrier molecule or solid support; R⁷is hydrogen, halogen, alkyl, substituted alkyl, alkoxy, substitutedalkoxy, hydroxyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, amino, substituted amino, alkylamino, substitutedalkylamino, aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether,sulfoalkyl, carboxyalkyl, aminoalkyl, reporter molecule, reactive group,carrier molecule or solid support; R⁸ is hydrogen; R⁹ is hydrogen; R¹⁰is hydrogen; R¹¹ is hydrogen; and R¹² is hydrogen.
 68. The kit accordingto claim 67, further comprising instructions for detecting the presenceor absence of lead, mercury, nickel, lanthanum or cadmium ions in asample.
 69. The kit according to claim 67, further comprisinginstructions for detecting the presence or absence of lead, mercury,nickel, lanthanum or cadmium ions in a sample using flow cytometry. 70.The kit according to claim 67, further comprising one or more of asample preparation reagent, a buffering agent, aqueous metal ionreporter molecule dilution buffer, an additional detection reagent, ametal ion calibration reagent, a positive control, a metal ion indicatorother than for lead, mercury, nickel, lanthanum or cadmium ions, anantibody or fragment thereof or a reference dye standard.
 71. The kitaccording to claim 67, wherein R is H, a salt ion or CH₂OC(O)CH₃. 72.The kit according to claim 67, wherein said R is hydrogen or a salt ion.73. The kit according to claim 67, wherein said R is CH₂OC(O)CH₃. 74.The kit according to claim 67, wherein at least one of R³, R⁶ and R⁷ isa reporter molecule.
 75. The kit according to claim 67, wherein R³ or R⁶is a reporter molecule.
 76. The kit according to claim 67, wherein thereporter molecule is a chromophore, fluorophore, fluorescent protein,phosphorescent dye or a tandem dye.
 77. The kit according to claim 67,wherein the reporter molecule is a xanthene, indole, cyanine, oxazole,dansyl, borapolyazaindacene, benzofuran, quinazolinone, benzazole,oxazine, pyrene, naphthalene, coumarin, biotin, enzyme substrate orfluorescent protein.
 78. The kit according to claim 77, wherein thexanthene is a fluorescein or derivative thereof, rhodamine or derivativethereof, rhodol or derivative thereof or rosamine or a derivativethereof
 79. The kit according to claim 77, wherein the reporter moleculeis independently substituted by hydrogen, halogen, amino, substitutedamino, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkoxy, sulfo, solid support, reactive group orcarrier molecule.
 80. The kit according to claim 77, wherein thereporter molecule is independently substituted by a lipophilic group.81. The kit according to claim 80, wherein the lipophilic group is an AMor acetate ester.
 82. The kit according to claim 67, wherein thereporter group, reactive group, solid support and carrier moleculecomprise a linker that is a single covalent bond, or a covalent linkagethat is linear or branched, cyclic or heterocyclic, saturated orunsaturated, having 1-20 nonhydrogen atoms selected from the groupconsisting of C, N, P, O and S; and are composed of any combination ofether, thioether, amine, ester, carboxamide, sulfonamide, hydrazidebonds and aromatic or heteroaromatic bonds.
 83. The kit according toclaim 67, wherein the reactive group is an acrylamide, an activatedester of a carboxylic acid, a carboxylic ester, an acyl azide, an acylnitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, anamine, an aryl halide, an azide, an aziridine, a boronate, adiazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine,an imido ester, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidite, a reactive platinum complex, a silyl halide, a sulfonylhalide, a thiol or a photoactivatable group.
 84. The kit according toclaim 67, wherein the reactive group is carboxylic acid, succinimidylester of a carboxylic acid, hydrazide, amine or a maleimide.
 85. The kitaccording to claim 67, wherein the carrier molecule is an amino acid, apeptide, a protein, a polysaccharide, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid polymer, a hapten, a psoralen, a drug, ahormone, a lipid, a lipid assembly, a synthetic polymer, a polymericmicroparticle, a biological cell or a virus.
 86. The kit according toclaim 67, wherein the carrier molecule is an antibody or fragmentthereof, an avidin or streptavidin, a biotin, a blood component protein,a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG bindingprotein, a fluorescent protein, a growth factor, a lectin, alipopolysaccharide, a microorganism, a metal binding protein, a metalchelating moiety, a non-biological microparticle, a peptide toxin, aphosphotidylserine-binding protein, a structural protein, asmall-molecule drug, or a tyramide.
 87. The kit according to claim 67,wherein the solid support is a microfluidic chip, a silicon chip, amicroscope slide, a microplate well, silica gels, polymeric membranes,particles, derivatized plastic films, glass beads, cotton, plasticbeads, alumina gels, polysaccharides, polyvinylchloride, polypropylene,polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, orsuperparamagnetic bead.
 88. The kit according to claim 67, wherein thesolid support is sepharose, poly(acrylate), polystyrene,poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch,FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose,diazocellulose or starch.
 89. The kit according to claim 67, wherein thecompound is

and a biologically compatible counterion.